Biomedical subjects

Shoshana Levy

Publications and source records attributed to Shoshana Levy.

At least 19 recordsLinked to original sources

PubMed2006

Cell-free production of scFv fusion proteins: an efficient approach for personalized lymphoma vaccines.

The unique immunoglobulin (Ig) idiotype on the surface of each B-cell lymphoma represents an ideal tumor-specific antigen for use as a therapeutic vaccine. We have used an Escherichia coli-based, cell-free protein-expression system to produce a vaccine within hours of cloning the Ig genes from a B-cell tumor. We demonstrated that a fusion protein consisting of an idiotypic single chain Fv antibody fragment (scFv) linked to a cytokine (GM-CSF) or to an immunostimulatory peptide was an effective lymphoma vaccine. These vaccines elicited humoral immune responses against the native Ig protein displayed on the surface of a tumor and protected mice against tumor challenge with efficacy equal to that of the conventional Ig produced in a mammalian cell and chemically coupled to keyhole limpet hemocyanin. The cell-free E coli system offers a platform for rapidly generating individualized vaccines, thereby allowing much more efficient application in the clinic.

Animals↗

PubMed2006

Expression of human CD81 differently affects host cell susceptibility to malaria sporozoites depending on the Plasmodium species.

Plasmodium sporozoites can enter host cells by two distinct pathways, either through disruption of the plasma membrane followed by parasite transmigration through cells, or by formation of a parasitophorous vacuole (PV) where the parasite further differentiates into a replicative exo-erythrocytic form (EEF). We now provide evidence that following invasion without PV formation, transmigrating Plasmodium falciparum and Plasmodium yoelii sporozoites can partially develop into EEFs inside hepatocarcinoma cell nuclei. We also found that rodent P. yoelii sporozoites can infect both mouse and human hepatocytes, while human P. falciparum sporozoites infect human but not mouse hepatocytes. We have previously reported that the host tetraspanin CD81 is required for PV formation by P. falciparum and P. yoelii sporozoites. Here we show that expression of human CD81 in CD81-knockout mouse hepatocytes is sufficient to confer susceptibility to P. yoelii but not P. falciparum sporozoite infection, showing that the narrow P. falciparum host tropism does not rely on CD81 only. Also, expression of CD81 in a human hepatocarcinoma cell line is sufficient to promote the formation of a PV by P. yoelii but not P. falciparum sporozoites. These results highlight critical differences between P. yoelii and P. falciparum sporozoite infection, and suggest that in addition to CD81, other molecules are specifically required for PV formation during infection by the human malaria parasite.

Animals↗

PubMed2006

Building of the tetraspanin web: distinct structural domains of CD81 function in different cellular compartments.

The tetraspanin web is composed of a network of tetraspanins and their partner proteins that facilitate cellular interactions and fusion events by an unknown mechanism. Our aim was to unravel the web partnership between the tetraspanin CD81 and CD19, a cell surface signaling molecule in B lymphocytes. We found that CD81 plays multiple roles in the processing, intracellular trafficking, and membrane functions of CD19. Surprisingly, these different roles are embodied in distinct CD81 domains, which function in the different cellular compartments: the N-terminal tail of CD81 has an effect on the glycosylation of CD19; the first transmembrane domain of CD81 is sufficient to support the exit of CD19 from the endoplasmic reticulum, although the large extracellular loop (LEL) of CD81 associates physically with CD19 early during biosynthesis; and finally, the TM2 and TM3 domains of CD81 play a role in the transmission of signals initiated upon engagement of the LEL. The participation of distinct CD81 domains in varied functions may explain the pleiotropic effects of CD81 within the tetraspanin web.

Animals↗

PubMed2005

Reduced fertility of female mice lacking CD81.

In somatic cells, the tetraspanins CD81 and CD9 associate with each other, with additional tetraspanins and with non-tetraspanin molecules to form proteolipidic complexes. Here we show that CD81 is expressed on the surface of oocytes where it associates with tetraspanin-enriched membrane structures. A major CD9 and CD81 partner, CD9P-1, is also expressed by oocytes. Deletion of CD81 gene in mice results in a 40% reduction of female fertility. In vitro insemination indicated that this infertility is due to a deficiency of oocytes to fuse with sperm. While the fertility of CD9-/- mice is severely but not completely impaired, double knock-out CD9-/- CD81-/- mice were completely infertile indicating that CD9 and CD81 play complementary roles in sperm-egg fusion. Finally, a fraction of CD9 was transferred from CD81-/- oocytes to sperm present in the perivitelline space indicating that the defect of fusion of CD81-/- oocytes does not result from an impaired initial gamete interaction.

Animals↗

PubMed2005

The CD9 tetraspanin is not required for the development of peripheral B cells or for humoral immunity.

The CD9 tetraspanin is known to be expressed at high levels on marginal zone (MZ) B cells, B-1 B cells, and plasma cells, and its expression is believed to be dependent on signals derived via Btk. In CD9 null mice, however, the development and survival of MZ B cells, B-1 B cells, and plasma cells all appear to be unaffected, and humoral immune responses to T-dependent and T-independent Ags are similar to those seen in wild-type littermate controls. In wild-type mice, CD9 levels may serve to distinguish between the presumed MZ precursor B cell population in the spleen and other IgD-expressing transitional B cells that express lower levels of CD21 and CD1d. These results suggest that CD9 is dispensable for B cell development and humoral immunity, but that this protein may serve as an additional marker for the presumed MZ precursor population of splenic B cells.

Animals↗

PubMed2005

Kinetics of HCV envelope proteins' interaction with CD81 large extracellular loop.

We used BIAcore to analyze the kinetics of interactions between CD81 and hepatitis C virus (HCV) envelope proteins. We immobilized different forms of HCV envelope proteins (E1E2, E2, and E2(661)) on the sensor and monitored their interaction with injected fusion proteins of CD81 large extracellular loop (CD81LEL) and glutathione-S-transferase (CD81LEL-GST) or maltose binding protein (CD81LEL-MBP). The difference between the GST and MBP fusion proteins was their multimeric and monomeric forms, respectively. The association rate constants between CD81LEL-GST or CD81LEL-MBP and the E1E2, E2 or E2(661) HCV envelope proteins were similar. However, the dissociation rate constants of CD81LEL-MBP were higher than those of CD81LEL-GST. Interestingly, the dissociation rate constant of CD81LEL-GST from E1E2 was much lower than from E2 or E2(661). The interaction between both forms of the CD81LEL fusion proteins and the HCV envelope proteins best-fitted the "heterogeneous ligand" model. This model implies that two kinds of interactions occur between envelope proteins and CD81LEL: one is strong, the other is weak. It also implies that the heterogeneity is likely due to the HCV envelope proteins, which are known to form non-covalently linked heterodimers and disulfide-linked aggregate.

Antigens, CD↗

PubMed2005

Expression of the human germinal center-associated lymphoma (HGAL) protein, a new marker of germinal center B-cell derivation.

We identified the human germinal center-associated lymphoma (HGAL) in gene-expression profiling studies of diffuse large B-cell lymphoma (DLBCL). The expression of HGAL correlated with survival in patients with DLBCL. The HGAL gene is the human homolog of M17, a mouse gene expressed specifically in normal germinal center (GC) B cells. We generated a monoclonal antibody against the HGAL protein and show that HGAL is expressed in the cytoplasm of GC lymphocytes and in lymphomas of GC derivation. Among 727 lymphomas tested by immunohistochemistry on tissue microarrays, HGAL staining was found in follicular lymphomas (103 of 107), Burkitt lymphomas (40 of 40), mediastinal large B lymphomas (7 of 8), and in DLBCLs (103 of 151). Most marginal zone lymphomas lacked HGAL staining. Lymphocyte-predominant Hodgkin lymphomas (12 of 17) and, surprisingly, classical Hodgkin lymphomas (78 of 107) were found to be positive. Hierarchical clustering of comparative immunohistologic results in DLBCLs demonstrates that the expression of HGAL is similar to 2 other GC-associated proteins, BCL6 and CD10, but different from 2 markers associated with a non-GC phenotype, MUM1/IRF4 and BCL2. The restricted expression and GC specificity of HGAL protein suggest that it may have an important role in the diagnosis of specific lymphomas, and, potentially in the identification of subtypes associated with different prognoses.

Animals↗

PubMed2005

The tetraspanin web modulates immune-signalling complexes.

The tetraspanin web represents a new concept of molecular interactions in the immune system. Whereas most surface immune-modulating molecules involve receptor-ligand interactions, tetraspanins associate with partner proteins and facilitate their lateral positioning in the membrane. Moreover, the same tetraspanin molecule can associate with different proteins depending on the cell type. Most importantly, members of this family tend to associate with each other, together with their partners, in membrane microdomains that provide a scaffold for the transmission of external stimuli to intracellular-signalling components.

Animals↗

PubMed2005

Protein-protein interactions in the tetraspanin web.

Tetraspanins are evolutionarily conserved membrane proteins that tend to associate laterally with one another and to cluster dynamically with numerous partner proteins in membrane microdomains. Consequently, members of this family are involved in the coordination of intracellular and intercellular processes, including signal transduction; cell proliferation, adhesion, and migration; cell fusion; and host-parasite interactions.

Animals↗

PubMed2004

Increased density of retinal pigment epithelium in cd81-/- mice.

Our laboratories have focused on the role of the tetraspanin CD81 in the regulation of mitotic activity. Previously we have shown that antibodies directed against CD81 can block the proliferation of cultured retinal pigment epithelial (RPE) cells. The present study investigates the role of this protein by analyzing the structure of the adult retina in mice with a null mutation of cd81. Adult cd81(-/-) mice were produced by crossing two inbred strains, NIHS-BC/Tac and 129X1/SvJ, carrying the cd81 mutation as heterozygotes (+/-). Seven cd81(-/-) mice and 11 wildtype (cd81(+/+)) littermates were anesthetized and perfused with paraformaldehyde. The eyes were removed and processed for examination by light and electron microscopy. In general, the retinas of the cd81(-/-) mice appeared normal. However, upon close examination, there was an 18% increase in the number of RPE nuclei in the cd81(-/-) mice. The photoreceptor layer of the cd81(-/-) mice was significantly thinner than that of the wild-type mice, even though there was no difference in the total thickness of the retinas in the two groups of mice. At the electron microscopic level we did not observe any differences in cell-cell junctions in the retinas of the cd81(-/-) mice as compared to their wild-type littermates. These data support a role for CD81 controlling cell-cycle and the number of RPE nuclei in the mouse retina.

Animals↗

PubMed2004

The tetraspanin CD81 is necessary for partitioning of coligated CD19/CD21-B cell antigen receptor complexes into signaling-active lipid rafts.

Tetraspanins have been hypothesized to facilitate the organization of functional multimolecular membrane complexes. In B cells the tetraspanin CD81 is a component of the CD19/CD21 complex. When coligated to the B cell Ag receptor (BCR), the CD19/CD21 complex significantly enhances BCR signaling in part by prolonging the association of the BCR with signaling-active lipid rafts. In this study CD81 is shown to associate with lipid rafts upon coligation of the BCR and the CD19/CD21 complex. Using B cells from CD81-deficient mice we demonstrate that in the absence of CD81, coligated BCR and CD19/CD21 complexes fail to partition into lipid rafts and enhance BCR signaling from rafts. Furthermore, a chimeric CD19 protein that associates only weakly if at all with CD81 fails to promote the association of coligated BCR with lipid rafts. The requirement for CD81 to promote lipid raft association may define a novel mechanism by which tetraspanins function as molecular facilitators of signaling receptors.

Adjuvants, Immunologic↗

PubMed2003

The tetraspanin CD81 regulates the expression of CD19 during B cell development in a postendoplasmic reticulum compartment.

CD81 is a widely expressed tetraspanin that associates in B cells with CD19 in the CD19-CD21-CD81 signaling complex. CD81 is necessary for normal CD19 expression; cd81(-/-) B cells express lower levels of CD19, especially cd81(-/-) small pre-BII cells, which are almost devoid of surface CD19. The dependence of CD19 expression on CD81 is specific to this particular tetraspanin since cd9(-/-) B cells express normal levels of CD19. Furthermore, expression of human CD81 in mouse cd81(-/-) B cells restored surface CD19 to normal levels. Quantitative analysis of CD19 mRNA demonstrated normal levels, even in cd81(-/-) pre-BII cells. Analysis of CD19 at the protein level identified two CD19 glycoforms in both wild-type and cd81(-/-) B cells. The higher M(r) glycoform is significantly reduced in cd81(-/-) B cells and is endoglycosidase H (endo-H) resistant. In contrast, the low M(r) glycoform is comparably expressed in cd81(-/-) and in wild-type B cells and is endo-H sensitive. Because endo-H sensitivity is tightly correlated with endoplasmic reticulum localization, we suggest that the dependency of CD19 expression on CD81 occurs in a postendoplasmic reticulum compartment where CD81 is necessary for normal trafficking or for surface membrane stability of CD19.

Animals↗

PubMed2003

The CD81 tetraspanin facilitates instantaneous leukocyte VLA-4 adhesion strengthening to vascular cell adhesion molecule 1 (VCAM-1) under shear flow.

Leukocyte integrins must rapidly strengthen their binding to target endothelial sites to arrest rolling adhesions under physiological shear flow. We demonstrate that the integrin-associated tetraspanin, CD81, regulates VLA-4 and VLA-5 adhesion strengthening in monocytes and primary murine B cells. CD81 strengthens multivalent VLA-4 contacts within subsecond integrin occupancy without altering intrinsic adhesive properties to low density ligand. CD81 facilitates both VLA-4-mediated leukocyte rolling and arrest on VCAM-1 under shear flow as well as VLA-5-dependent adhesion to fibronectin during short stationary contacts. CD81 also augments VLA-4 avidity enhancement induced by either chemokine-stimulated Gi proteins or by protein kinase C activation, although it is not required for Gi protein or protein kinase C signaling activities. In contrast to other proadhesive integrin-associated proteins, CD81-promoted integrin adhesiveness does not require its own ligand occupancy or ligation. These results provide the first demonstration of an integrin-associated transmembranal protein that facilitates instantaneous multivalent integrin occupancy events that promote leukocyte adhesion to an endothelial ligand under shear flow.

Animals↗

PubMed2003

Hepatitis C virus (HCV) and lymphomagenesis.

Hepatitis C virus (HCV) is the major cause for non-A, non-B hepatitis. Most HCV-infected individuals do not clear the virus resulting in a chronic infection that may potentially lead to liver cirrhosis and hepatocellular carcinoma. In addition to hepatic manifestations, HCV infection is associated with B cell lymphoproliferative disorders, including mixed cryoglobulinemia, usually a benign condition, and overt B cell lymphoma. A direct role of HCV infection in the genesis of these B cell lymphoproliferative disorders has been suggested initially by epidemiological studies and is supported by recent studies, which analyzed the monoclonal B cells that proliferate in these disorders. How HCV induces B cell lymphoproliferative disorders is still unclear, it is probably not due to direct change of phenotype in B cells after viral infection, but may be due to an HCV-antigen driven process. Support for this hypothesis comes from the analysis of monoclonal B cells found in these disorders, which use a restricted repertoire of immunoglobulin variable region genes that are similar to those used by B cells that secrete anti-HCV antibodies. The fact that monoclonal IgM is resolved in HCV-infected patients who responded to anti-viral treatment supports the linkage between antigen persistence and B cell proliferation. Finally, the linkage between benign B cell proliferation and overt lymphoma is supported by the identification of a pre-malignant B cell clone that subsequently converted to an overt B cell lymphoma. The molecular basis for viral induced B cell proliferation is still unknown. One possibility is that HCV stimulates the proliferation of monoclonal B cells via their HCV-specific B cell receptor (BCR) on the cell surface. Binding of the HCVenvelope proteins to a cellular ligand, CD81, may also enhance this antigen-driven process. A recent report on regression of splenic marginal zone lymphoma after anti-viral treatment with interferon and ribavirin has significantly strengthened the cause-effect relationship between HCV infection and lymphoma. Further studies should determine whether BCRs expressed on HCV-associated lymphomas, particularly those that regress in response to anti-viral therapy, bind HCV antigens that stimulate their proliferation.

B-Lymphocytes↗

PubMed2003

CD81-dependent binding of hepatitis C virus E1E2 heterodimers.

Hepatitis C virus (HCV) is the leading cause of chronic liver disease worldwide. HCV is also the major cause of mixed cryoglobulinemia, a B-lymphocyte proliferative disorder. Direct experimentation with native viral proteins is not feasible. Truncated versions of recombinant E2 envelope proteins, used as surrogates for viral particles, were shown to bind specifically to human CD81. However, truncated E2 may not fully mimic the surface of HCV virions because the virus encodes two envelope glycoproteins that associate with each other as E1E2 heterodimers. Here we show that E1E2 complexes efficiently bind to CD81 whereas truncated E2 is a weak binder, suggesting that truncated E2 is probably not the best tool with which to study cellular interactions. To gain better insight into virus-cell interactions, we developed a method by which to isolate E1E2 complexes that are properly folded. We demonstrate that purified E1E2 heterodimers bind to cells in a CD81-dependent manner. Furthermore, engagement of B cells by purified E1E2 heterodimers results in their aggregation and in protein tyrosine phosphorylation, a hallmark of B-cell activation. These studies provide a possible clue to the etiology of HCV-associated B-cell lymphoproliferative diseases. They also delineate a method by which to isolate biologically functional E1E2 complexes for the study of virus-host cell interaction in other cell types.

Antigens, CD↗

PubMed2003

Recognition of native hepatitis C virus E1E2 heterodimers by a human monoclonal antibody.

The majority of hepatitis C virus (HCV)-infected individuals progress from acute to chronic disease, despite the presence of a strong humoral immune response to the envelope glycoproteins E1 and E2. When expressed in mammalian cells, E1 and E2 form both noncovalently linked E1E2 heterodimers, believed to be properly folded, and disulfide-linked, high-molecular-weight aggregates that are misfolded. Previously, we identified 10 human monoclonal antibodies (HMAbs) that bind E2 glycoproteins from different genotypes. Here we demonstrate that one of these HMAbs, CBH-2, is unique in its ability to distinguish between properly folded and misfolded envelope proteins. This HMAb recognizes HCV-E2 only when complexed with E1. The E1E2 complexes recognized by CBH-2 are noncovalently linked heterodimers and not misfolded disulfide-linked, high-molecular-weight aggregates. The E1E2 heterodimers seen by CBH-2 no longer associate with the endoplasmic reticulum chaperone calnexin and are likely to represent the prebudding form of the HCV virion.

Antibodies, Monoclonal↗

PubMed2002

Expression of human CD81 in transgenic mice does not confer susceptibility to hepatitis C virus infection.

We previously demonstrated that hepatitis C virus (HCV) binds to human CD81 through the E2 glycoprotein. Therefore, expression of the human CD81 molecule in transgenic mice was expected to provide a new tool to study HCV infection in vivo, as the chimpanzee is the only species currently available as a laboratory animal model that can be infected with HCV. We produced transgenic mice expressing the human CD81 protein in a wide variety of tissues. We confirmed binding of recombinant E2 glycoprotein to the liver tissue as well as to thymocytes and splenic lymphocytes in the transgenic mice. We inoculated chimpanzee plasma infected with HCV into these animals. None of these transgenic animals showed evidence of viral replication. Furthermore, human CD81 transgenic mice that lack expression of endogenous mouse CD81 were also resistant to HCV infection. We conclude that expression of human CD81 alone is insufficient to confer susceptibility to HCV infection in the mouse. The presence of additional possible factors for HCV infection is discussed.

Animals↗

PubMed2002

Hepatocyte CD81 is required for Plasmodium falciparum and Plasmodium yoelii sporozoite infectivity.

Plasmodium sporozoites are transmitted through the bite of infected mosquitoes and first invade the liver of the mammalian host, as an obligatory step of the life cycle of the malaria parasite. Within hepatocytes, Plasmodium sporozoites reside in a membrane-bound vacuole, where they differentiate into exoerythrocytic forms and merozoites that subsequently infect erythrocytes and cause the malaria disease. Plasmodium sporozoite targeting to the liver is mediated by the specific binding of major sporozoite surface proteins, the circumsporozoite protein and the thrombospondin-related anonymous protein, to glycosaminoglycans on the hepatocyte surface. Still, the molecular mechanisms underlying sporozoite entry and differentiation within hepatocytes are largely unknown. Here we show that the tetraspanin CD81, a putative receptor for hepatitis C virus, is required on hepatocytes for human Plasmodium falciparum and rodent Plasmodium yoelii sporozoite infectivity. P. yoelii sporozoites fail to infect CD81-deficient mouse hepatocytes, in vivo and in vitro, and antibodies against mouse and human CD81 inhibit in vitro the hepatic development of P. yoelii and P. falciparum, respectively. We further demonstrate that the requirement for CD81 is linked to sporozoite entry into hepatocytes by formation of a parasitophorous vacuole, which is essential for parasite differentiation into exoerythrocytic forms.

Animals↗