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Biomedical subjects

R S Fujinami

Publications and source records attributed to R S Fujinami.

At least 73 records · Page 4Linked to original sources
PubMed1988

Monoclonal antibody defines determinant between Theiler's virus and lipid-like structures.

Theiler's murine encephalomyelitis virus is known to cause a chronic demyelinating disease in mice. The contributions of immunologic factors, i.e. humoral and cellular responses to virus and/or myelin components, and direct virus-cell interactions leading to demyelination are still unclear. One important factor could be antibody initiation of myelin destruction. Here we describe four monoclonal antibodies that react with Theiler's murine encephalomyelitis virus. Three of these neutralize the virus and one of these three could also bind to various lipid-like structures including galactocerebroside, a myelin component. Further, this monoclonal antibody reacted with oligodendrocyte-like cells in vitro. All four monoclonal antibodies reacted with VP-1 by Western blot analysis. Thus, an immune response generated by virus that cross-reacts with a myelin element such as galactocerebroside could play a role in directing autoimmune processes toward myelin destruction.

Animals↗
PubMed1988

Sequence homology and immunologic cross-reactivity of human cytomegalovirus with HLA-DR beta chain: a means for graft rejection and immunosuppression.

A peptide (Leu-Gly-Arg-Pro-Asp-Glu-Asp-Ser-Ser-Ser-Ser-Ser-Ser-Ser-Cys) that was identical to residues 82 through 96 of a predicted protein of 208 amino acids from the immediate-early region (IE-2) nucleic acid sequence of human cytomegalovirus was chemically synthesized. By computer analysis, the first five amino acids of this peptide showed sequence homology to the beta chain of the human histocompatibility complex HLA-DR. The homologous amino acids, 53 through 57, were located in a region that is conserved between the human DR beta chain and the beta chain of the H-2 class II histocompatibility antigen for mice. The shared region between the IE-2 protein and DR beta chain were similar in both hydrophilicity and predicted beta-turn potential. The IE-2 viral peptide induced antibodies that specifically recognized the human DR beta chain. These observations describe a protein encoded by the IE-2 region of human cytomegalovirus that contains sequence homology and shows immunologic cross-reactivity with a conserved domain of HLA-DR and suggest a mechanism to explain how human cytomegalovirus infection contributes to graft rejection after transplantation.

Amino Acid Sequence↗
PubMed1988

Theiler's virus infection in nude mice: viral RNA in vascular endothelial cells.

Infection of athymic (nu/nu) mice with Theiler's murine encephalomyelitis virus results in an acute encephalitis which resembles poliomyelitis. Immunohistochemistry and in situ hybridization were used to delineate the presence of viral proteins and RNA in the nervous systems of nude mice infected with the Daniels strain of Theiler's virus. This system permits the analysis of a viral infection in the absence of an effective immune response. By immunohistochemistry, viral antigen was found in the processes and cell bodies of neurons and glial cells. Besides the presence of viral antigen in these cell types, by in situ hybridization, Theiler's virus RNA was also found in cells associated with vascular endothelium in the brains and spinal cords of these infected mice. Theiler's virus RNA-positive endothelial cells were observed not only near the primary lesions but also away from demonstrable lesions in normal-appearing regions in the central nervous system. Earlier work had suggested an intra-axonal dissemination for this virus (M. C. Dal Canto and H. L. Lipton, Am. J. Pathol. 106:20-29, 1982). Our findings are consistent with this model but also suggest an additional mechanism for virus spread within the central nervous system, i.e., by infecting vascular cells and crossing the blood-brain barrier. Lastly, after Theiler's murine encephalomyelitis virus infection, not only glial cells but also endothelial cells express major histocompatibility complex class II (la) antigen on their surface (M. Rodriguez, M. L. Pierce, and E. A. Howie, J. Immunol. 138:3438-3442, 1987). Our demonstration of Theiler's virus-infected endotheliumlike cells may explain interactions of virus products in stimulating antigen presentation.

Animals↗
PubMed1987

Effects of regional spinal X-irradiation on demyelinating disease caused by Theiler's virus, mouse hepatitis virus or experimental allergic encephalomyelitis.

The effects of X-irradiation on the course of chronic demyelinating disease were examined in mice with experimental allergic encephalitis (EAE), mouse hepatitis virus (MHV) or Theiler's virus (DAV) infection. One month after the induction of EAE or 2-16 months after inoculation of DAV, exposure of the cervical spinal cord to 20 Gy X-rays caused local exacerbation of disease activity but spinal irradiation did not affect MHV-induced demyelination. In EAE, there was a significant increase in the number of inflammatory cells in the irradiated part of the cord. Mice infected with DAV showed locally increased demyelination and axonal degeneration but no change in the titer of infectious virus within the cord. Thus in DAV infection, as in EAE, the exacerbation of disease seemed to be due to vascular or immunological factors rather than viral reactivation.

Animals↗
PubMed1987

Characterization of in vitro transcription and transcriptional products of measles virus.

Ribonucleoprotein complexes isolated from measles virus-infected HeLa cells contained an RNA-dependent RNA polymerase activity that catalyzed the incorporation of ribonucleotides into ribonucleic acid. The ribonucleoprotein complexes were composed of measles virus nucleoprotein, phosphoprotein, and a large protein, as well as viral RNA. The kinetics of RNA synthesis at different temperatures, time intervals, and protein, ribonucleotide, and mono- and divalent cation concentrations were analyzed. Enzyme activity was maximum at 4 h at 25 degrees C in the presence of 100 mM Na+-2.5 mM Mg2+-1 mM ribonucleotides. Actinomycin D and alpha-amanitin had no effect on the enzyme activity. Addition of cytoplasmic extracts from uninfected HeLa cells to the reaction mixture did not increase the incorporation of ribonucleotides into RNA. The in vitro synthesized RNAs were characterize by slot blot analysis and quantitated by densitometer scanning. All mRNAs coding for the structural proteins of measles virus were synthesized. Nucleoprotein RNA was the most abundant species made, followed by phosphoprotein, hemagglutinin, fusion protein, matrix protein, and large-protein RNAs. The system described here resulted in the first efficient transcription of measles virus RNA and analysis of products.

Amanitins↗
PubMed1986

Viruses disrupt functions of human lymphocytes. II. Measles virus suppresses antibody production by acting on B lymphocytes.

Measles virus infection is associated with suppression of immune functions both in vivo and in vitro. The virus infects T lymphocytes, B lymphocytes, and monocytes, but does not produce cytolysis. One consequence of infection in vitro is the failure of T and B lymphocyte mixtures to cooperate in secreting Ig in a PWM-driven system. Here we report that this defect in Ig secretion resides in the infected B lymphocyte, but not in the T lymphocyte or monocyte. Further, NK cells are not involved, since neither their depletion nor reconstitution abrogates suppression of B cell function. Proliferation of B cells in the early culture period is suppressed, suggesting that measles virus suppresses B cell development at the activation or proliferation stages, but does not affect terminal differentiation into Ig secreting cells.

Antibody Formation↗
PubMed1986

Mechanism of Theiler's virus-induced demyelination in nude mice.

In its natural murine host, infection with Theiler's murine encephalomyelitis virus (TMEV) produces a chronic, progressive demyelinating disease. To help elucidate the role of host immune mechanisms involved in demyelination, we studied TMEV infection in Nude mice. These animals demonstrated rising titers of infectious virus within the central nervous system and failed to produce anti-TMEV antibody. Neurologic signs including the development of severe hind limb paralysis were evident approximately 2 weeks postinfection with most animals succumbing within the first month. Immunoperoxidase studies demonstrated viral antigen in the cytoplasm of neurons and glial cells for the entire period of observation. Plaques of demyelination associated with scanty inflammatory infiltrates were present in the spinal cord by 14 days postinfection. Electron microscopic studies of the involved white matter revealed numerous degenerating glial cells, many of which contained paracrystalline arrays of picornavirus within their cytoplasm. Some of the infected glial cells were identified as oligodendrocytes by demonstrating their myelin-plasma membrane connections. The studies indicate that in Nude mice TMEV causes a lytic infection of oligodendrocytes producing demyelination independent of the T lymphocyte immune system.

Animals↗
PubMed1985

Amino acid homology between the encephalitogenic site of myelin basic protein and virus: mechanism for autoimmunity.

Amino acid sequence homology was found between viral and host encephalitogenic protein. Immune responses were then generated in rabbits by using the viral peptide that cross-reacts with the self protein. Mononuclear cell infiltration was observed in the central nervous systems of animals immunized with the viral peptide. Myelin basic protein (MBP) is a host protein whose encephalitogenic site of ten amino acids induces experimental allergic encephalomyelitis. By computer analysis, hepatitis B virus polymerase (HBVP) was found to share six consecutive amino acids with the encephalitogenic site of rabbit MBP. Rabbits given injections of a selected eight- or ten-amino acid peptide from HBVP made antibody that reacted with the predetermined sequences of HBVP and also with native MBP. Peripheral blood mononuclear cells from the immunized rabbits proliferated when incubated with either MBP or HBVP. Central nervous system tissue taken from these rabbits had a histologic picture reminiscent of experimental allergic encephalomyelitis. Thus, viral infection may trigger the production of antibodies and mononuclear cells that cross-react with self proteins by a mechanism termed molecular mimicry. Tissue injury from the resultant autoallergic event can take place in the absence of the infectious virus that initiated the immune response.

Amino Acid Sequence↗
PubMed1984

Antigenic modulation induced by monoclonal antibodies: antibodies to measles virus hemagglutinin alters expression of other viral polypeptides in infected cells.

Polyclonal antibody to measles virus can have profound effects on external (outer plasma membrane) as well as internal (cytoplasmic) viral polypeptides expressed in infected cells. The process, termed "antibody-induced antigenic modulation," was further investigated by using monoclonal antibody to several viral polypeptides. Four monoclonal antibodies against the viral hemagglutinin had the ability to decrease the expression of the phosphoprotein, fusion, and membrane protein. A monoclonal antibody to the nucleocapsid protein did not cause these changes. The observed decreases were not due to preferential degradation of viral polypeptides as determined by pulse-chase experiments. Our results indicate that a specific signal to an epitope on the plasma membrane (monoclonal antibody measles virus hemagglutinin) can alter the expression of measles virus phosphoprotein and membrane protein, both polypeptides present in the cytoplasm of infected cells.

Antibodies, Bacterial↗
PubMed1983

Mapping of the major histocompatibility complex and viral antigens on the plasma membrane of a measles virus-infected cell.

The two measles virus glycoproteins, the hemagglutinin and fusion protein, are expressed on the surfaces of infected cells. Although the two molecules are chemically distinct, they associate on the cell surface, judging from their ability to comigrate (co-cap). However, neither is directly complexed with the major histocompatibility (MHC) gene products, HLA-A, -B, -C or -D, on the plasma membrane, based on results from three distinct assays. First, in tests of capping, these viral glycoproteins failed to comigrate with any HLA determinant. Second, electron microscopy showed that the viral glycoproteins occupied domains on the plasma membrane distinct from MHC gene products; 125I labeling of cell surface determinants and subsequent analysis by immune precipitation and PAGE confirmed this result. Third, incubation of measles virus-infected cells in the presence of monoclonal or polyclonal antibodies to measles virus glycoproteins removed the viral glycoproteins from the cells' surfaces but did not cause a corresponding decrease in amounts of HLA molecules. These results indicate that the hemagglutinin and fusion polypeptides of measles virus lie in close association on the plasma membrane; however, neither is linked with MHC gene products.

Antigens, Viral↗
PubMed1983

Molecular mimicry in virus infection: crossreaction of measles virus phosphoprotein or of herpes simplex virus protein with human intermediate filaments.

Using monoclonal antibodies, we demonstrate that the phosphoprotein of measles virus and a protein of herpes simplex virus type 1 crossreact with an intermediate filament protein of human cells. This intermediate filament protein, probably vimentin, has a molecular weight of 52,000, whereas the molecular weights of the measles viral phosphoprotein and the herpes virus protein are 70,000 and 146,000, respectively. Crossreactivity was shown by immunofluorescent staining of infected and uninfected cells and by immunoblotting. The monoclonal antibody against measles virus phosphoprotein did not react with herpes simplex virus protein and vice versa, indicating that these monoclonal antibodies recognize different antigenic determinants on the intermediate filament molecule. The significance of these results in explaining the appearance of autoantibodies during virus infections in humans is discussed.

Antibodies, Monoclonal↗
PubMed1983

Serologic relatedness between Thy-1.2 and actin revealed by monoclonal antibody.

Monoclonal antibodies with affinity for Thy-1.2 on thymocytes also can bind to actin within marsupial, murine, and human cells. A similar cross-reactivity between Thy-1.1 and vimentin was revealed by Dulbecco and co-workers employing monoclonals. A computer-assisted analysis of the amino acid composition provided suggestive evidence for the occurrence of sequence homology between Thy-1.1 or Thy-1.2, actin, and vimentin that likely accounts for the serologic relatedness detected by hybridoma antibodies.

Actins↗
PubMed1983

Infection with vaccinia favors the selection of hybridomas synthesizing autoantibodies against intermediate filaments, one of them cross-reacting with the virus hemagglutinin.

Immunization of mice with lysates from infected cells and pure vaccinia virus generated hybridomas, a large fraction of which make autoantibodies binding to intermediate filaments (greater than IF) and vimentin. Antibodies that can be detected in preimmune mice as weakly binding to the cytoskeleton (greater than CS) are elevated in potency after immunization with either active or UV-inactivated pure vaccinia, suggesting that virus replication may not be obligatory for activation of greater than CS lymphocyte clones. Demonstration of a cross-reactivity of one IF monoclonal antibody with virally coded hemagglutinin, expressed at the surface, raises questions about the development of pathologic conditions associated with virus-mediated autoimmunity.

Animals↗
PubMed1981

Failure to cleave measles virus fusion protein in lymphoid cells.

The host-directed cleavage of measles virus fusion protein on infected lymphoid cells was studied to understand the mechanism of viral persistence in lymphoid cells in vivo. Several lymphoblastoid cell lines were infected with measles virus, and the viral glycoproteins expressed on the cell's surface were radiolabeled and analyzed for cleavage of fusion (F(0)) to F(1) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Daudi and Ramos lymphoblastoid cells were deficient in their ability to cleave measles virus fusion protein and correspondingly produced low titers of infectious measles virus, Daudi cells being more defective than Ramos cells. In contrast, other lymphoblastoid cells studied, Victor, Raji, Wi-L2, RPMI 8866, and Seraphine, cleaved the fusion polypeptide and made significantly more infectious virus. Despite their defect in cleaving F protein, Daudi cells were able to assemble and release (noninfectious) measles virus particles into the fluid phase. The deficit in Daudi cells was corrected by fusing infected Daudi cells with cleavage-competent cells such as Victor or Raji. Furthermore, the cleavage event performed by competent cells could be mimicked at the plasma membrane by treating infected Daudi cells with trypsin, implicating the role of a plasma membrane enzyme in cleaving F(0) to F(1) during measles virus infection. Hence, lymphoid cells deficient in the plasma membrane enzyme required to cleave F protein are permissive for measles virus, maintain viral gene products, produce mostly noninfectious virus, and fail to place the biologic activity F(1) protein on their surfaces.

Antigens, Surface↗
PubMed1981

Purification of measles virus glycoproteins and their integration into artificial lipid membranes.

We report a simple method for the isolation of the measles virus glycoproteins, and their subsequent incorporation into artificial lipid bilayers. The two viral glycoproteins, HA and F, were isolated in preparative amounts from disrupted purified virus by lentil lectin affinity chromatography. The proteins were reconstituted into single bilayer lipid vesicles by: (i) exchanging the non-dialysable detergent Nonidet P40 (NP40) for a dialysable one, octylglucoside, while the proteins were immobilized on the lectin column and (ii) co-dialysis of the eluted glycoproteins in octylglucoside with phosphatidylcholine. The resultant 'virosomes' had visible 'spikes' and possessed haemagglutinating activity. These measles virosomes should provide a useful reagent for studying immune responses to measles virus, independent of the immunosuppressive effects of the whole virus.

Centrifugation, Density Gradient↗