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D R Shafren

Publications and source records attributed to D R Shafren.

At least 19 recordsLinked to original sources
PubMed2000

The integrins alpha3beta1 and alpha6beta1 physically and functionally associate with CD36 in human melanoma cells. Requirement for the extracellular domain OF CD36.

Lateral association between different transmembrane glycoproteins can serve to modulate integrin function. Here we characterize a physical association between the integrins alpha(3)beta(1) and alpha(6)beta(1) and CD36 on the surface of melanoma cells and show that ectopic expression of CD36 by CD36-negative MV3 melanoma cells increases their haptotactic migration on extracellular matrix components. The association was demonstrated by co-immunoprecipitation, reimmunoprecipitation, and immunoblotting of surface-labeled cells lysed in Brij 96 detergent. Confocal microscopy illustrated the co-association of alpha(3) and CD36 in cell membrane projections and ruffles. A requirement for the extracellular domain of CD36 in this association was shown by co-immunoprecipitation experiments using surface-labeled MV3 melanoma or COS-7 cells that had been transiently transfected with chimeric constructs between CD36 and intercellular adhesion molecule 1 (ICAM-1) or with a truncation mutant of CD36. CD36 is known to engage in signal transduction and to localize to membrane microdomains or rafts in several cell types. Toward a mechanistic explanation for the functional effects of CD36 expression, we demonstrate that in fractionated Triton X-100 lysates of the MV3 cells stably transfected with CD36, CD36 was greatly enriched with the detergent-insoluble fractions that represent plasma membrane rafts. Significantly, when these fractionated lysates were reprobed for endogenous beta(1) integrin, it was found that a 4-fold increase in the proportion of the mature protein was contained within the detergent-insoluble fractions when extracted from the CD36-transfected cells compared with MV3 cells transfected with vector only. These results suggest that in melanoma cells CD36 expression may induce the sequestration of certain integrins into membrane microdomains and promote cell migration.

Animals↗
PubMed2000

Cytoplasmic interactions between decay-accelerating factor and intercellular adhesion molecule-1 are not required for coxsackievirus A21 cell infection.

Coxsackievirus A21 (CAV-21) employs a cell receptor complex of decay-accelerating factor (DAF) and intercellular adhesion molecule-1 (ICAM-1) for cell infectivity. In this study, the nature of potential extra- and/or intracellular interactions between DAF and ICAM-1 involved in picornaviral cell entry was investigated. Firstly, it was shown that intracellular interplay between DAF and ICAM-1 is not required for CAV-21 infection, as CAV-21 lytic infection mediated via the DAF/ICAM-1 receptor complex is not inhibited by replacement of the transmembrane and cytoplasmic domains of ICAM-1 with those from an unrelated cell surface molecule, CD36. By immunoprecipitation, chemical cross-linking and picornaviral binding assays, the existence of a close spatial association between DAF and ICAM-1 on the surface of ICAM-1-transfected RD cells was confirmed. Furthermore, it was shown that potential extracellular DAF/ICAM-1 interactions are likely to occur in an area on or proximal to DAF SCR3 and may influence the route of CAV-21 cell entry.

Animals↗
PubMed1999

Picornavirus receptor down-regulation by plasminogen activator inhibitor type 2.

Therapeutic interference with virus-cell surface receptor interactions represents a viable antiviral strategy. Here we demonstrate that cytoplasmic expression of the serine protease inhibitor (serpin), plasminogen activator inhibitor type 2 (PAI-2), affords a high level of protection from lytic infection by multiple human picornaviruses. The antiviral action of PAI-2 was mediated primarily through transcriptional down-regulation of the following virus receptors: intercellular adhesion molecule 1 (ICAM-1, a cellular receptor for the major group of rhinoviruses), decay-accelerating factor (a cellular receptor for echoviruses and coxsackieviruses), and to a lesser extent the coxsackie-adenovirus receptor protein (a cellular receptor for group B coxsackieviruses and group C adenoviruses). Expression of related cell surface receptors, including membrane cofactor protein and the poliovirus receptor, remained unaffected. These findings suggest that PAI-2 and/or related serpins may form the basis of novel antiviral strategies against picornavirus infections and also therapeutic interventions against ICAM-1-mediated respiratory inflammation.

CD55 Antigens↗
PubMed1998

Antibody binding to individual short consensus repeats of decay-accelerating factor enhances enterovirus cell attachment and infectivity.

Decay-accelerating factor (DAF), a widely expressed membrane complement-regulatory protein, is utilized as a cellular receptor by many human enteric pathogens. We show here that the binding of two enteroviruses to individual short consensus repeats (SCR) of DAF on the cell surface is greatly augmented by mAb binding to an alternate SCR: Coxsackievirus A21 binding to the SCR1 of DAF is increased by Ab binding to SCR3 and, conversely, Echovirus 7 binding to SCR3 is enhanced severalfold by Ab binding to SCR1. These Ab-induced increases in viral binding also resulted in increased viral infectivity. Using purified soluble DAF in a solid phase assay it was found that Ab binding to SCR1 is increased greatly in the presence of an Ab against SCR3 and, reciprocally, Ab against SCR1 greatly increases Ab binding to SCR3. In contrast to the results obtained with the larger viral particles, however, this reciprocal Ab-induced enhancement of binding is not seen when measuring Ab binding to membrane-bound DAF SCR on the cell surface. These findings provide a possible explanation for functional differences between membrane-bound and soluble DAF with implications for a potential role for DAF-binding molecules in regulating DAF function. This is the first demonstration of enhancement of viral infectivity mediated by Ab against the viral receptor.

Animals↗
PubMed1998

Viral cell entry induced by cross-linked decay-accelerating factor.

Decay-accelerating factor (DAF) mediates cellular attachment for many human picornaviruses. In most cases, viral binding to DAF is itself insufficient to permit cell infectivity, with a second, functional internalization receptor being required to facilitate this process. Previously, we postulated that the role of DAF in enterovirus cell infection is as a sequestration receptor, maintaining a reservoir of bound virus in an infectious state, awaiting interaction with functional internalization receptors. Many of these functional receptors possess the capacity to induce relatively rapid changes in capsid conformations, resulting in the formation of altered particles (A-type particles). In this report, we show that antibody-cross-linked DAF, in contrast to endogenous surface-expressed forms, can act as a functional virus receptor to mediate coxsackie A21 virus (CAV21) lytic cell infection. In contrast to the situation with ICAM-1-mediated CAV21 infection, in which high levels of A-type particles are formed, cross-linked DAF-induced CAV21 replication occurs in the absence of detectable A-particle formation.

Binding Sites↗
PubMed1997

Integrin alpha v beta 6 enhances coxsackievirus B1 lytic infection of human colon cancer cells.

Viral entry into host cells depends upon specific interactions between virus attachment proteins and cell surface receptors that enable virus binding and internalization of virus and/or the virus-receptor complex. We have recently reported that the ubiquitous cell surface molecule, decay-accelerating factor (DAF), is a major cell attachment receptor for Coxsackieviruses B1, B3, and B5. However, DAF permits only virus binding and not virus internalization, invoking the presence of secondary or accessory receptors. Among the known receptors for enteroviruses are members of the cell adhesion molecule family known as integrins. In the present study, we found that expression of the epithelial-restricted integrin, alpha v beta 6, on colonic epithelial cells significantly enhanced Coxsackievirus B1-mediated cell lysis. Importantly, the viral-mediated cell killing required the presence of the 11-amino-acid C-terminal cytoplasmic extension unique to the beta 6 subunit, providing the first evidence of regulation of viral infectivity by integrin cytoplasmic domains. These results indicate that alpha v beta 6 expression on intestinal epithelial cells critically affects Coxsackievirus B1 infectivity. This may be essential in the conversion of asymptomatic enterovirus infection into clinically apparent disease.

Antigens, Neoplasm↗
PubMed1997

CD36 forms covalently associated dimers and multimers in platelets and transfected COS-7 cells.

CD36 is a transmembrane glycoprotein expressed on the surface of a number of cell types. The analysis of CD36 from platelets using immunoblotting, gel filtration, and native PAGE indicated the presence of high molecular complexes exceeding the Mr of monomeric CD36. Experiments using transfected COS-7 cells revealed these complexes were homodimers and -multimers of CD36. The multimers could be dissociated by treatment with a reducing agent, indicating they were formed by intermolecular cysteine-bridging. Mutagenesis of the cDNA for CD36 implicated the cysteines in the extracellular domain of the molecule. The potential physiological roles of CD36 multimerisation are discussed.

Animals↗
PubMed1997

Mouse cells expressing human intercellular adhesion molecule-1 are susceptible to infection by coxsackievirus A21.

Competitive viral binding assays have revealed previously that coxsackievirus A21 (CAV21) and human rhinovirus 14 (HRV14) share a common cell surface receptor. More recently, intercellular adhesion molecule-1 (ICAM-1) has been identified as the cellular receptor for HRV-14. Also, anti-ICAM-1 monoclonal antibodies (MAbs) blocked infection by HRV14, CAV13, CAV18, and CAV21, suggesting that these viruses share this receptor; however, this has never been established by more direct methods. In this study we show conclusively that CAV21 binds to ICAM-1 and that MAbs directed against the N-terminal domain of the molecule inhibit this attachment. Furthermore, we show that the specific interaction between ICAM-1 and 160S CAV21 virions induces formation of 135S A particles. Finally, we show transfection of normally nonsusceptible mouse L cells with human ICAM-1 cDNA renders them susceptible to infection by CAV21.

Animals↗
PubMed1997

A decay-accelerating factor-binding strain of coxsackievirus B3 requires the coxsackievirus-adenovirus receptor protein to mediate lytic infection of rhabdomyosarcoma cells.

The composition of the cellular receptor complex for coxsackievirus B3 (CVB3) has been an area of much contention for the last 30 years. Recently, two individual components of a putative CVB3 cellular receptor complex have been identified as (i) decay-accelerating factor (DAF) and (ii) the coxsackievirus-adenovirus receptor protein (CAR). The present study elucidates the individual roles of DAF and CAR in cell entry of CVB3 Nancy. First, we confirm that the DAF-binding phenotype of CVB3 correlates to the presence of key amino acids located in the viral capsid protein, VP2. Second, using antibody blockade, we show that complete protection of permissive cells from infection by high input multiplicities of CVB3 requires a combination of both anti-DAF and anti-CAR antibodies. Finally, it is shown that expression of the CAR protein on the surface of nonpermissive DAF-expressing RD cells renders them highly susceptible to CVB3-mediated lytic infection. Therefore, although the majority of CVB3 Nancy attaches to the cell via DAF, only virus directly interacting with the CAR protein mediates lytic infection. The role of DAF in CVB3 cell infection may be analogous to that recently described for coxsackievirus A21 (D. R. Shafren, D. J. Dorahy, R. A. Ingham, G. F. Burns, and R. D. Barry, J. Virol. 71:4736-4743, 1997), in that DAF may act as a CVB3 sequestration site, enhancing viral presentation to the functional CAR protein.

Cell Line↗
PubMed1997

Coxsackievirus A21 binds to decay-accelerating factor but requires intercellular adhesion molecule 1 for cell entry.

It is becoming increasingly apparent that many viruses employ multiple receptor molecules in their cell entry mechanisms. The human enterovirus coxsackievirus A21 (CAV21) has been reported to bind to the N-terminal domain of intercellular adhesion molecule 1 (ICAM-1) and undergo limited replication in ICAM-1-expressing murine L cells. In this study, we show that in addition to binding to ICAM-1, CAV21 binds to the first short consensus repeat (SCR) of decay-accelerating factor (DAF). Dual antibody blockade using both anti-ICAM-1 (domain 1) and anti-DAF (SCR1) monoclonal antibodies (MAbs) is required to completely abolish binding and replication of high-titered CAV21. However, the binding of CAV21 to DAF, unlike that to ICAM-1, does not initiate a productive cell infection. The capacity of an anti-DAF (SCR3) MAb to block CAV21 infection but not binding, coupled with immunoprecipitation data from chemical cross-linking studies, indicates that DAF and ICAM-1 are closely associated on the cell surface. It is therefore suggested that DAF may function as a low-affinity attachment receptor either enhancing viral presentation or providing a viral sequestration site for subsequent high-affinity binding to ICAM-1.

Animals↗
PubMed1996

CD36 is spatially associated with glycoprotein IIb-IIIa (alpha IIb beta 3) on the surface of resting platelets.

Platelet activation and aggregation induced by agonists such as thrombin are accompanied by the phosphorylation of several proteins on tyrosine. Such tyrosine phosphorylation is dependent upon activation and ligand engagement of the major integrin receptor on the surface of platelets, glycoprotein (GP) IIb-IIIa (alpha IIb beta 3), but how this is accomplished is not known. The only platelet membrane GP known to associated with non receptor tyrosine kinases is CD36 (GPIV) which forms associations with pp60Fyn, pp62Yes, and pp54/58Lyn, and antibodies directed against CD36 activate platelets in a process dependent upon GPIIb-IIIa. These and other data suggest a physical association between the two membrane GPs, IIb-IIIa and CD36. By the use of immunoprecipitation of lysates of platelets that have been surface labeled and chemically crosslinked we show here that CD36 and GPIIb-IIIa are spatially associated on the surface of resting platelets.

Antibodies, Monoclonal↗
PubMed1996

A common epitope on platelet integrin alpha IIb beta 3 (glycoprotein IIbIIIa; CD41b/CD61) and alpha M beta 2 (Mac-1; CDIIb/CD18) detected by a monoclonal antibody.

Evidence is presented that the mAb 25E11, directed against the platelet integrin alpha IIb beta 3 (glycoprotein IIbIIIa;CD41b/CD61) also binds the distinct myeloid cell integrin alpha M beta 2 (Mac-1;CDIIb/CD18). The Ab is shown to identify only the alpha IIb beta 3 integrin complex and not the individual subunits in crossed Ab immunoelectrophoresis of platelet lysate. From cultured human macrophages, sequential immunoprecipitation of labeled glycoproteins indicated that 25E11 also bound the Mac-1 (CD11b/CD18) complex. This was confirmed using COS-7 and WOP cells doubly transfected with alpha M (CD11b) and beta 2 (CD18) or with alpha L (CD11a) and beta 2 when it was found that the Ab bound only the alpha M beta 2 transfectants. Studies with these cells and the RC2A myeloid cell line stimulated with tetradecanoyl phorbol acetate or FMLP indicated that the 25E11 epitope on Mac-1 did not depend on cell activation for its expression. The rationale for this cross-reactivity is not known, but since the 25E11 Ab inhibits the function of both platelets and myeloid cells, it is likely that this shared epitope is important to integrin function. Given the expression of this epitope on IIbIIIa and Mac-1, the dominant integrins of platelets and granulocyte/macrophage cells, but not on other integrins, a role of this epitope in the early events of inflammation is suggested.

Antibodies, Monoclonal↗
PubMed1995

Coxsackieviruses B1, B3, and B5 use decay accelerating factor as a receptor for cell attachment.

Receptor binding and subsequent cell-mediated internalization or disassembly are the initial steps in virus replication. Cell surface molecules that participate in this process are the primary determinants of virus tissue tropism. Monoclonal antibody blockade, immunoprecipitation, and DNA transfection were used to identify decay accelerating factor as a major cell attachment receptor for coxsackieviruses B1, B3, and B5. However, expression of human decay acceleration factor on the surface of nonpermissive murine fibroblasts led only to virus attachment without subsequent replication, and it was concluded that an additional cellular cofactor(s) is required to facilitate cell entry and subsequent replication.

Antibodies, Monoclonal↗
PubMed1992

Antibody responses to avian encephalomyelitis virus vaccines when administered by different routes.

Antibody responses to a commercial avian encephalomyelitis virus (AEV) vaccine administered by different routes were measured by an enzyme-linked immunosorbent assay (ELISA). Responses to single doses of vaccine administered by the ocular route to 10% of a flock were comparable with those obtained when all birds received a single dose in the drinking water. However, ocular vaccination of 5% of the flock resulted in significantly lower responses than those obtained when 10% were vaccinated. Maternal antibody was shown by the ELISA to persist in chickens from vaccinated flocks for up to 21 days after hatching. Day-old chickens with serum absorbances of < 0.3 at 492 nm, as determined by the ELISA, were shown to be susceptible to intracerebral challenge with the neurotropic Van Roekel strain of AEV.

Administration, Oral↗
PubMed1991

Pathogenesis of avian encephalomyelitis viruses.

The pathogenesis of a field strain, a vaccine strain and the egg-adapted Van Roekel strain of avian encephalomyelitis virus in susceptible chicken embryos and day-old chickens was investigated using enzyme-linked immunosorbent assays for the detection of virus-specific antibody and antigen. The Van Roekel strain was shown to be highly neurotropic whereas the field and vaccine strains were enterotropic. Radioimmuno-precipitation studies using Na125I-labelled purified virus failed to detect any differences in the composition of the structural viral proteins of each strain that could account for these differences. As expected, the field and vaccine strains were more efficient than the Van Roekel strain at inducing antibody following oral administration. Primary cultures of chicken embryo brain cells supported the growth of the Van Roekel strain to a much greater extent than the field and vaccine strains.

Animals↗
PubMed1990

Development and application of an improved infectivity assay for the standardization of avian encephalomyelitis vaccines.

An improved infectivity assay for avian encephalomyelitis viruses (AEVs) is described in which susceptible 7-day-old chicken embryos were inoculated with dilutions of a particular AEV strain and, after incubation for 12 days, the presence of viral antigen in brains was detected by an enzyme-linked immunosorbent assay (ELISA). The assay (the brain antigen ELISA), which may be used for the titration of both egg-adapted and non-egg-adapted strains, can be completed within 12 days. For the conventional essays of non-egg-adapted vaccine strains, embryos are infected in ovo and hatched chickens examined for neurological signs after 3-4 weeks. No animal handling facilities are required with the brain antigen ELISA and results of comparable sensitivity to the hatch-out assay were obtained.

Animals↗
PubMed1989

Development and application of an ELISA technique for the detection of antibody to avian encephalomyelitis viruses.

A rapid sensitive enzyme-linked immunosorbent assay for the detection of antibody to avian encephalomyelitis viruses (AEVs) in chickens using purified antigen is described. The procedure differed from others which have been described for AEV, in that it involved a negative antigen subtraction step which accounted for the variable adhesiveness of chicken sera to plastic surfaces. The procedure was reproducible (between-assay coefficient of variation 8.95 per cent) and a good correlation was observed with results obtained by neutralisation index tests (r = 0.91, P less than 0.1). The assay detects only AEV-specific antibody and allows monitoring of the spread of AEV in flocks.

Animals↗