Anti-Transferrin Receptor Antibody [MEM-189] (A85952)

Equine rhinitis A virus (ERAV) is genetically closely related to foot-and-mouth disease virus (FMDV), and both are now classified within the genus Aphthovirus of the family Picornaviridae. For disease security reasons, FMDV can be handled only in high-containment facilities, but these constraints do not apply to ERAV, making it an attractive alternative for the study of aphthovirus biology. Here, we show, using immunofluorescence, pharmacological agents, and dominant negative inhibitors, that ERAV entry occurs (as for FMDV) via clathrin-mediated endocytosis and acidification of early endosomes. This validates the use of ERAV as a model system to study the mechanism of cell entry by FMDV.

CD1 proteins are a family of major histocompatibility complex (MHC) class I-like antigen-presenting molecules that present lipids to T cells. The cytoplasmic tails (CTs) of all human CD1 isoforms, with the exception of CD1a, contain tyrosine-based sorting motifs, responsible for the internalization of proteins by the clathrin-mediated pathway. The role of the CD1a CT, which does not possess any sorting motifs, as well as its mode of internalization are not known. We investigated the internalization and recycling pathways followed by CD1a and the role of its CT. We found that CD1a can be internalized by a clathrin- and dynamin-independent pathway and that it follows a Rab22a- and ADP ribosylation factor (ARF)6-dependent recycling pathway, similar to other cargo internalized independent of clathrin. We also found that the CD1a CT is S-acylated. However, this posttranslational modification does not determine the rate of internalization or recycling of the protein or its localization to detergent-resistant membrane microdomains (DRMs) where we found CD1a to be enriched. We also show that plasma membrane DRMs are essential for efficient CD1a-mediated antigen presentation. These findings place CD1a closer to MHC class I in its trafficking and potential antigen-loading compartments among CD1 isoforms. Furthermore, we identify CD1a as a new marker for the clathrin- and dynamin-independent and DRM-dependent pathway of internalization as well as the Rab22a- and ARF6-dependent recycling pathway.

Activated tyrosine kinases have been frequently implicated in the pathogenesis of cancer, including acute myeloid leukemia (AML), and are validated targets for therapeutic intervention with small-molecule kinase inhibitors. To identify novel activated tyrosine kinases in AML, we used a discovery platform consisting of immunoaffinity profiling coupled to mass spectrometry that identifies large numbers of tyrosine-phosphorylated proteins, including active kinases. This method revealed the presence of an activated colony-stimulating factor 1 receptor (CSF1R) kinase in the acute megakaryoblastic leukemia (AMKL) cell line MKPL-1. Further studies using siRNA and a small-molecule inhibitor showed that CSF1R is essential for the growth and survival of MKPL-1 cells. DNA sequence analysis of cDNA generated by 5'RACE from CSF1R coding sequences identified a novel fusion of the RNA binding motif 6 (RBM6) gene to CSF1R gene generated presumably by a t(3;5)(p21;q33) translocation. Expression of the RBM6-CSF1R fusion protein conferred interleukin-3 (IL-3)-independent growth in BaF3 cells, and induces a myeloid proliferative disease (MPD) with features of megakaryoblastic leukemia in a murine transplant model. These findings identify a novel potential therapeutic target in leukemogenesis, and demonstrate the utility of phosphoproteomic strategies for discovery of tyrosine kinase alleles.

BACKGROUND AND OBJECTIVE:

Development of miniaturized imaging systems with molecular probes enables examination of molecular changes leading to initiation and progression of colorectal cancer in an azoxymethane (AOM)-induced mouse model of the disease. Through improved and novel studies of animal disease models, more effective diagnostic and treatment strategies may be developed for clinical translation. We introduce use of a miniaturized multimodal endoscope with lavage-delivered fluorescent probes to examine dynamic microenvironment changes in an AOM-treated mouse model.

STUDY DESIGN/MATERIALS AND METHODS:

The endoscope is equipped with optical coherence tomography (OCT) and laser induced fluorescence (LIF) imaging modalities. It is used with Cy5.5-conjugated antibodies to create time-resolved molecular maps of colon carcinogenesis. We monitored in vivo changes in molecular expression over a five month period for four biomarkers: epithelial growth factor receptor (EGFR), transferrin receptor (TfR), transforming growth factor beta 1 (TGFβ1), and chemokine (C-X-C motif) receptor 2 (CXCR2). In vivo OCT and LIF images were compared over multiple time points to correlate increases in biomarker expression with adenoma development.

RESULTS:

This system is uniquely capable of tracking in vivo changes in molecular expression over time. Increased expression of the biomarker panel corresponded to sites of disease and offered predictive utility in highlighting sites of disease prior to detectable structural changes. Biomarker expression also tended to increase with higher tumor burden and growth rate in the colon.

CONCLUSION:

We can use miniaturized dual modality endoscopes with fluorescent probes to study the tumor microenvironment in developmental animal models of cancer and supplement findings from biopsy and tissue harvesting.

© 2014 Wiley Periodicals, Inc.

Previous studies have shown that iron redistribution and deposition in the brain occurs in some neurodegenerative diseases, and oxidative damage due to abnormal iron level is a primary cause of neuronal death. In the present study, we used the single prolonged stress (SPS) model to mimic post-traumatic stress disorder (PTSD), and examined whether iron was involved in the progression of PTSD. The anxiety-like behaviors of the SPS group were assessed by the elevated plus maze (EPM) and open field tests, and iron levels were measured by inductively coupled plasma optical emission spectrometer (ICP-OES). Expression of glucocorticoid receptors and transferrin receptor 1 (TfR1) and ferritin (Fn) was detected by Western blot and immunohistochemistry in selected brain areas; TfR1 and Fn mRNA expression were detected by quantitative-polymerase chain reaction (Q-PCR). Ultrastructures of the hippocampus were observed under a transmission electron microscope. Our results showed that SPS exposure induced anxiety-like symptoms and increased the level of serum cortisol and the concentration of iron in key brain areas such as the hippocampus, prefrontal cortex, and striatum. The stress induced region-specific changes in both protein and mRNA levels of TfR1 and Fn. Moreover, swelling mitochondria and cell apoptosis were observed in neurons in brain regions with iron accumulation. We concluded that SPS stress increased iron in some cognition-related brain regions and subsequently cause neuronal injury, indicating that the iron may function in the pathology of PTSD.

Glucocorticoids are used extensively to treat autoimmune hemolytic anemias. Some beneficial effects of glucocorticoid pulse therapy have also been reported in sickle cell disease and paroxysmal nocturnal hemoglobinuria. Based on established concepts of hemoglobin (Hb) toxicity and physiologic Hb scavenger systems, we evaluated whether glucocorticoids could support an adaptive response to extracellular Hb independently of their immunosuppressive activities. Using global proteome and transcriptome analysis with mass-spectrometry (isobaric tag for relative and absolute quantitation and liquid chromatography-mass spectrometry) and gene-array experiments, we found that glucocorticoid treatment in vitro and in patients on glucocorticoid-pulse therapy polarized monocytes into a M2/alternatively activated phenotype with high Hb-scavenger receptor (CD163) expression and enhanced Hb-clearance and detoxification capability. Monocytes concurrently exposed to the interactive activity of glucocorticoids and extracellular Hb were characterized by high expression of a group of antioxidant enzymes known to be regulated by the conserved oxidative response transcription factor nuclear factor E2-related factor. Further, suppressed transferrin receptor, together with high ferroportin expression, pointed to a shift in iron homeostasis directed toward an increased cellular export of heme-derived iron. Therefore, stimulating Hb-endocytosis by CD163 and enhancing antioxidative homeostasis and iron recycling may be an essential activity of glucocorticoids that helps alleviate the adverse effects of extracellular Hb.

This review paper describes a new technology, mass cytometry, that addresses applications typically run by flow cytometer analyzers, but extends the capability to highly multiparametric analysis. The detection technology is based on atomic mass spectrometry. It offers quantitation, specificity and dynamic range of mass spectrometry in a format that is familiar to flow cytometry practitioners. The mass cytometer does not require compensation, allowing the application of statistical techniques; this has been impossible given the constraints of fluorescence noise with traditional cytometry instruments. Instead of "colors" the mass cytometer "reads" the stable isotope tags attached to antibodies using metal-chelating labeling reagents. Because there are many available stable isotopes, and the mass spectrometer provides exquisite resolution between detection channels, many parameters can be measured as easily as one. For example, in a single tube the technique allows for the ready detection and characterization of the major cell subsets in blood or bone marrow. Here we describe mass cytometric immunophenotyping of human leukemia cell lines and leukemia patient samples, differential cell analysis of normal peripheral and umbilical cord blood; intracellular protein identification and metal-encoded bead arrays.

BACKGROUND AIMS:

Cell-based gene therapy is an alternative to viral and non-viral gene therapy. Emerging evidence suggests that mesenchymal stem cells (MSC) are able to migrate to sites of tissue injury and have immunosuppressive properties that may be useful in targeted gene therapy for sustained specific tissue engraftment.

METHODS:

In this study, we injected intravenously (i.v.) 1x10(6) MSC, isolated from green fluorescent protein (GFP) transgenic rats, into Rif-1 fibrosarcoma-bearing C3H/HeN mice. The MSC had been infected using a lentiviral vector to express stably the luciferase reporter gene (MSC-GFP-luci). An in vivo imaging system (IVIS 200) and Western blotting techniques were used to detect the distribution of MSC-GFP-luci in tumor-bearing animals.

RESULTS:

We observed that xenogenic MSC selectively migrated to the tumor site, proliferated and expressed the exogenous gene in subcutaneous fibrosarcoma transplants. No MSC distribution was detected in other organs, such as the liver, spleen, colon and kidney. We further showed that the FGF2/FGFR pathways may play a role in the directional movement of MSC to the Rif-1 fibrosarcoma. We performed in vitro co-culture and in vivo tumor growth analysis, showing that MSC did not affect the proliferation of Rif-1 cells and fibrosarcoma growth compared with an untreated control group. Finally, we demonstrated that the xenogenic MSC stably expressing inducible nitric oxide synthase (iNOS) protein transferred by a lentivirus-based system had a significant inhibitory effect on the growth of Rif-1 tumors compared with MSC alone and the non-treatment control group.

CONCLUSIONS:

iNOS delivered by genetically modified iNOS-MSC showed a significant anti-tumor effect both in vitro and in vivo. MSC may be used as a target gene delivery vehicle for the treatment of fibrosarcoma and other tumors.