The Roles of Membrane Rafts in CD32A-Mediated Phagocytosis

Membrane rafts are highly dynamic heterogeneous sterol- and sphingolipid-rich micro-domains on cell surfaces. They are generally believed to provide residency for cell surface molecules (e.g., adhesion and signaling molecules) and scaffolding to facilitate the functions of these molecules such as membrane trafficking, receptor transport, cell signaling, and endocytosis.
The governing, or overall hypothesis, for this project is that membrane rafts provide residency for Fc[gamma]RIIA (CD32A) on K562 cells, and that by doing so they provide a platform from which Fc[gamma]RIIA initiate or carry out their functions, which include migration, signaling, phagocytic synapse formation, and internalization of IgG opsonized targets.
Using immuno-fluorescent laser scanning confocal microscopy and reflection interference microscopy (RIM), we studied the spatial and temporal distributions of membrane rafts and surface receptors, signaling molecules, and cell organelles during the formation of phagocytic contact areas. K562 cells, which naturally express CD32A, a cell surface receptor for the Fc portion of Immuno-globulin G(IgG), was chosen as a model for neutrophils. An opsonized target was modeled using a glass supported lipid bilayer reconstituted with IgG. CD32A was found to cluster and co-localize with membrane rafts. Placing the K562 cells on the lipid bilayer triggered a process of contact area formation that includes binding between receptors and ligands, their recruitment to the contact area, a concurrent membrane raft movement to and concentration in the contact area, and transport of CD32A, IgG, and membrane rafts to the Golgi complex. Characterization of these processes was performed using agents known to disrupt detergent resistant membranes (DRMs), dissolve actin microfilaments, and inhibit myosin motor activity, which abolished the CD32A clusters and prevented the contact area formation. 
The relevance to phagocytosis of contact area formation between K562 cells and lipid bilayers was demonstrated using micro-beads coated with a lipid bilayer reconstituted with IgG as the opsonized target instead of the glass supported planar lipid bilayer. Disruption of membrane rafts, salvation of the actin cytoskeleton, and inhibition of myosin II activity were found to inhibit phagocytosis.
These data suggest membrane rafts play several important roles in CD32A mediated phagocytosis including pre-clustering CD32A, transport of CD32A to the phagocytic cup, and transport of the opsonized target towards the Golgi complex. Here we have provided evidence that membrane rafts serve as platforms which are used to cluster CD32A and transport CD32A along the actin cytoskeleton to the site of phagocytic synapse formation thus allowing for the quick assembly of a phagocytic synapse.
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Effects of Anchor Structure and Glycosylation of Fcγ Receptor III on Ligand Binding Affinity

Isoforms of the Fcγ receptor III (FcγRIII or CD16) are cell surface receptors for the Fc portion of IgG and important regulators of humoral immune responses. Different ligand binding kinetics of FcγRIII isoforms are obtained in three dimensions by surface plasmon resonance and in two dimensions by a micropipette adhesion frequency assay. We show that the anchor structure of CD16 isoforms isolated from the cell membrane affects their binding affinities in a ligand-specific manner. Changing the receptor anchor structure from full to partial to none decreases the ligand binding affinity for human IgG1 (hIgG1) but increases it for murine IgG2a (mIgG2a). Removing N-glycosylation from the CD16 protein core by tunicamycin also increases the ligand binding affinity. Molecular dynamics simulations indicate that deglycosylation at Asn-163 of CD16 removes the steric hindrance for the CD16-hIgG1 Fc binding and thus increases the binding affinity. These results highlight an unexpected sensitivity of ligand binding to the receptor anchor structure and glycosylation and suggest their respective roles in controlling allosterically the conformation of the ligand binding pocket of CD16

Immune Complex-Induced, Nitric Oxide-Mediated Vascular Endothelial Cell Death by Phagocytes Is Prevented with Decoy FcyReceptors

Autoimmune vasculitis is an endothelial inflammatory disease that results from the deposition of immune-complexes (ICs) in blood vessels. The interaction between Fcgamma receptors (FcyRs) expressed on inflammatory cells with ICs is known to cause blood vessel damage. Hence, blocking the interaction of ICs and inflammatory cells is essential to prevent the IC-mediated blood vessel damage. Thus we tested if uncoupling the interaction of FcyRs and ICs prevents endothelium damage. Herein, we demonstrate that dimeric FcyR-Igs prevented nitric oxide (NO) mediated apoptosis of human umbilical vein endothelial cells (HUVECs) in an in vitro vasculitis model. Dimeric FcyR-Igs significantly inhibited the IC-induced upregulation of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) release by murine monocytic cell line. However, FcyR-Igs did not affect the exogenously added NO-induced upregulation of pro-apoptotic genes such as Bax (15 fold), Bak (35 fold), cytochrome-C (11 fold) and caspase-3 (30 fold) in HUVECs. In conclusion, these data suggest that IC-induced NO could be one of the major inflammatory mediator promoting blood vessel inflammation and endothelial cell death during IC-mediated vasculitis which can be effectively blocked by dimeric decoy FcyRs

Mucosal Delivery of Particulate Breast Cancer Vaccine

Vaccination has been widely used as a mode of protection against various diseases by taking advantage of host\u27s immune system. Even though vaccination has provided relief from many infectious diseases, vaccination for cancer still remains a challenge. Cancer is caused by mutated cell functioning leading to uncontrolled growth in the organ of genesis and further possible metastasis worsens the situation. In spite of various current therapies such as surgery, chemotherapy and radiation therapy, we are still lacking behind in the race with this evolving disease. There are two major approaches for vaccination: prophylactic or therapeutic. Prophylactic vaccines find their applications in the prevention of viral, bacterial, or parasitic infectious diseases such as influenza, HIV, tuberculosis, malaria, pneumonia, polio, small pox, etc., which are caused by foreign antigens. However, in the case of cancer, which is caused by mutated self-cells, vaccine formulation is a challenging task as it requires immune response against self-cell antigens without causing auto-immune response

Immuno-Oncology: The Past, the Present and the Future

Keynote speaker for 2019 GA Research Day

Comparative structural and lectin-binding studies on γ-glutamyltransferase from human adult liver, fetal liver and primary hepatoma

γ-Glutamyltransferase was purified to apparent homogeneity from human adult liver, fetal liver and hepatoma by deoxycholate extraction, immunoaffinity chromatography, papain digestion, phenyl-Sepharose chromatography and preparative polyacrylamide gel electrophoresis. The purified enzyme from all three sources had an apparent M<SUB>r</SUB> of 82 000 by Sephadex G-150 gel filtration and on dodecyl sulphate/polyacrylamide gel electrophoresis two nonidentical subunits of M<SUB>r</SUB> 57000 and 23000 were obtained. The pI of all three forms was 3.85 and after neuraminidase treatment they each gave at least five bands with pI values ranging over 5.9-6.6. Sialic acid content was 188 (adult liver), 182 (fetal liver) and 188 (hepatoma) nmol/mg protein. Total neutral sugar content was 702 (adult and fetal liver) and 700 (hepatoma) nmol/mg protein. The hexosamine content of the enzyme from all the three sources was the same (354 nmol/mg protein) and galactosamine was absent. Partially purified hydrophobic and hydrophilic forms of γ-glutamyltransferase from all the three sources were precipitated by Concanavalin A, Ricinus communis agglutinin and wheat germ agglutinin. These results show that γ-glutamyltransferase from human adult liver, fetal liver and hepatoma are structurally similar and that the elevated levels found in fetuses and hepatoma are only a quantitative increase and are not due to a new isoenzyme

Custom designing therapeutic cancer vaccines. Delivery of immunostimulatory molecule adjuvants by protein transfer

Attempts to create vaccines for humans against invading pathogens such as viruses and bacteria have met with tremendous success. The process of developing vaccines against these pathogens is greatly aided by the fact that they contain antigens that are entirely foreign to humans. Although the knowledge and strategies developed for designing vaccines against these microbes may be of use in developing cancer vaccines, the poor antigenicity and immunosuppressive ability of cancers pose major hurdles to vaccine development. Established tumors have not only withstood immune screening and selection pressure, making them poor stimulators of an immune response, but have also adapted mechanisms to continue evading immune surveillance by creating an immunosuppressive environment. Also, genetic differences in immune responses to an antigen among individuals result in an antigenic profile that varies from patient to patient. Cancers bear such great similarities to normal cells in the body that, on a molecular level, the differences between cancerous and non-cancerous cells are minor. Therefore, developing vaccines which use the host\u27s own tumor tissues carries the risk of breaking tolerance to self-antigens that are present in the tumor tissue. Vaccination strategies that will optimally stimulate the immune system against tumor specific antigens under immunosuppressive conditions need to be developed. In practical terms, this calls for a method by which therapeutic vaccines may be custom-designed to treat cancers case by case. Ex vivo manipulation of dendritic cells and gene transfer of immunostimulatory molecules in ex vivo expanded tumors are being tested in both experimental models and also in human clinical trials. Some of them have met with limited success. Emerging technologies such as protein transfer, which make it possible to express immunostimulatory molecules on tumor cell membranes, offer the means to develop efficient tumor vaccines that are simple and fast, while being easy to store and administer in human patients. Progress in these techniques will move the cancer vaccine field a step closer towards realizing custom designed cancer vaccines in human clinical settings. © 2008 Landes Bioscience

"The creeping tumor:" An unusual presentation of upper urinary tract malignancy

Upper urinary tract urothelial malignancy accounts for 5-10% of urothelial carcinomas. Synchronous bladder carcinoma occurs in 2-4% of patients with upper urinary tract tumors. Urothelial malignancy involving the entire upper urinary tract is an extremely rare entity. Most upper urinary tract malignancies are transitional cell carcinomas (TCC), of which the sarcomatoid variant is very rare. These tumors pose a challenge to the radiologist. We herein report a case of TCC involving the entire collecting system of the left kidney, extending down along the ureter and projecting as a mass in the bladder