Inhibition of human lymphocyte function by organic solvents.

UNLABELLED: We studied the direct effect of reactive hydroxyl precursors and inhibitors on CD4+ T-cell function. We used hydrogen peroxide plus ferrous chloride as the hydroxyl radical-generating system and di-methyl sulphourea, di-methyl sulfoxide, pyrrolidine dithiocarbonate, methanol, and ethanol, at a noncytotoxic concentration, as inhibitors. The immune parameter studies were proliferation and interleukin-2 production by peripheral blood lymphocytes stimulated with anti-CD3 antibody, phytohemagglutinin and alloantigens; proliferation, interleukin-2 production and mRNA expression of interleukin-4 and interferon gamma by allogeneic CD4+ T-cell clones stimulated with alloantigens. The results show that lymphocytes produce significant amounts of reactive oxygen species as measured by malondialdehyde produced in cultures. The hydroxyl radical-generating system did not change any of the cellular responses studied although it doubled Malondialdehyde production. Hydroxyl radical scavengers significantly inhibited all responses at doses that didn't significantly decrease malondialdehyde production. DNA analysis failed to show evidence for apoptosis. CONCLUSION: Hydroxyl radical scavengers inhibit lymphocyte mitogenesis by a process that is independent of scavenging hydroxyl radicals.

Purification and characterization of Epstein-Barr virus gp340/220 produced by a bovine papillomavirus virus expression vector system.

Our initial results with a bovine papilloma virus (BPV) vector expression system indicated that we could produce significant amounts of Epstein-Barr virus (EBV) gp340/220 in the supernatant of a mouse fibroblast cell line. We have now extended these findings to show that the truncated version of gp340/220, where the membrane anchor sequence is deleted, is produced even after extended passage of the cells, at a level of approximately 1 mg/4 x 10(8) cells. A simple purification protocol using Sephacryl S300HR and gelatin agarose gives a product which is greater than 90% pure. This product is recognized by anti-gp340 monoclonal antibodies from five different epitope groups and induces antibody that recognizes the authentic gp340/220 and neutralizes EBV in vitro. The purified gp340/220 can be used in ELISA and stimulates the proliferation of T-cell clones specific for gp340/220. These characteristics, together with the fact that BPV-transformed lines have been utilized for the production of pharmaceuticals for use in humans, suggest that this gp340/220 is suitable as a source of antigen for vaccination to prevent EBV infection and related diseases.

Morphology of astroglia in colony cultures following transient exposure to potassium ion, hypoösmolarity and vasopressin.

Brain swelling is the major cause of delayed neuronal damage following injury to the central nervous system. Swelling of mouse astroglial cells was studied in colony cultures by light and electron microscopy. Swelling of suspended astroglial cells was studied by flow cytometry. Swelling caused by hypoösmolarity solution was more pronounced than that caused by 15 or 60 mM K+. Under both conditions swelling in both immature and mature astroglia was followed by a regulatory volume decrease. Arginine vasopressin caused mild astroglial swelling and atrial natriuretic peptide did not significantly affect cell volume. All changes in extracellular environment were associated with changes in the morphology of microvilli and varying amounts of membrane ruffling. Immature cells exhibited a delayed response to the application of atrial natriuretic peptide and less membrane ruffling following exposure to 60 mM K+ than mature astroglia. These nonspecific morphological changes are likely associated with changes in membrane ion pump activity.

Modified tetrazolium-based colorimetric method for determining the activities of anti-HIV compounds.

A modified tetrazolium-based colorimetric assay was used to determine the anti-HIV activities of ddAzThd, ddCyd, ddIno, and PFA. In this assay, poly-1-lysine-coated plates were used to attach the MT-2 cells to the bottom of the plates. A fixed amount of virus (50 TCID50) was used in each well. A modified version of the formula published by Pauwels et al. (1988) was used for calculating the percentage cell protection from virus infection. Using CC10/EC90 to calculate the selective indices, the decreasing order of selectivity against HIV-1 strain A87SF, was: ddAzThd greater than PFA greater than ddCyd greater than ddIno. Against HIV-1 strain A79SK-1 the decreasing order of selectivity was: PFA greater than ddIno greater than AzThd greater than ddCyd. The modified formula showed lack of anti-HIV activity for thymidine at non-toxic concentrations.

Cyclosporin abrogates virus-specific T-cell control of EBV-induced B-cell lymphoproliferation.

Renal and cardiac recipients undergoing aggressive immunosuppressive therapy with cyclosporin A have been reported to have unusually high incidences of Epstein-Barr virus (EBV) genome-positive lymphomas. T cells have been shown to be of critical importance in controlling the lymphoproliferative potential of the EB virus. EBV-specific T cell clones were generated in vitro by repeated antigenic restimulation in the presence of interleukin-2 (IL-2). These virus-specific cells were used to study cyclosporin A's effect on their activation, proliferation and cytotoxic function, and the possible abrogation of the cell's ability to control virus-induced lymphoproliferation. In this study, we show that cyclosporin A does not interfere with antigen recognition, since the T cell cytotoxic potential is unchanged. However, cyclosporin A induces a dose-dependent reduction in membrane IL-2 receptor expression which consequently limits the proliferation of the antigen-activated cell. This may translate in vivo to an insufficient expansion of the cytotoxic T cells which controls the outgrowth of the EBV-infected B cell.

Antigenic analysis of the Epstein-Barr virus major membrane antigen (gp350/220) expressed in yeast and mammalian cells: implications for the development of a subunit vaccine.

The Epstein-Barr virus (EBV) major surface membrane antigen, gp350/220, was expressed in recombinant yeast cells and in several recombinant mammalian cell lines. Each of the expressed proteins was analyzed for its ability to bind to a panel of anti-gp350/220 monoclonal antibodies and to a series of anti-EBV positive human sera. The antigens also were used as immunogens for the immunization of rabbits. Each expressed protein was found to be unique both in its pattern of reactivity to the various antibodies and in the spectrum of antibody induced following animal immunization. These results suggest that cell-specific post-translational modifications critically influence the antigenic presentation of the expressed proteins. Nonetheless, all of the mammalian cell-derived versions of the membrane antigen were found capable of inducing EBV-specific neutralizing antibodies.

Characterization of a major virion envelope glycoprotein complex of murine cytomegalovirus and its immunological cross-reactivity with human cytomegalovirus.

Three glycoproteins on the murine cytomegalovirus (MCMV) virion with apparent molecular weights of 150K (gp 150), 105K (gp 105), and 52K (gp52) were immunoprecipitated by two monoclonal antibodies (MAbs) 8G5.12A and 2E.12A. However, only 8G5.12A was able to neutralize MCMV infectivity in the presence of complement. The accessibility of these three glycoproteins to radiolabeling by surface-iodination reactions suggested that they were exposed on the surface of the virion. Western blot analysis of the three glycoproteins showed that gp150 shared antigenic determinants with gp105 and gp52. Briefly, the MAb 8G5.12A reacted with gp150 and gp105, whereas the MAb 2E8.12A reacted with gp150 and gp52. A third MAb 3H2.12A was also found to be reactive with gp150 and gp105 in Western blots, but was unable to immunoprecipitate these glycoproteins. Data from pluse-chase experiments suggested that all three virion glycoproteins were synthesized from a common 128K precursor, providing a partial explanation of their antigenic relatedness. Furthermore, we have demonstrated the presence of high-molecular-weight complexes formed by disulfide bonding between gp150, gp105, and gp52. Lastly, the MAb 8G5.12A was able to immunoprecipitate 84K and 99-110K glycoproteins from human CMV-infected WI-38 cells, demonstrating that conserved determinants exist between murine and human CMV envelope glycoproteins.

A neutralizing monoclonal antibody recognizes an 87K envelope glycoprotein on the murine cytomegalovirus virion.

An 87K glycoprotein (gp87) on the murine cytomegalovirus (MCMV) virion was immunoprecipitated by the neutralizing monoclonal antibody (MAb) 8D1.11A. The 87K glycoprotein is also radiolabeled in a surface iodination reaction, suggesting that it is exposed on the surface of the virion. Using a nondenaturing system of polyacrylamide gel electrophoresis in combination with Western blotting, we have shown that the epitope recognized by the MAb 8D1.11A resides on gp87. The failure of 8D1.11A to react with gp87 in a reduced and denatured form suggests that the epitope is recognized only when disulfide linkages are preserved. Our data also indicated that gp87 is present in the MCMV virion both in a monomeric form and as a component of disulfide-linked complexes. Using a two-dimensional gel electrophoresis system, we have demonstrated the presence of disulfide linkages between gp87 and virion polypeptides with apparent molecular weights of 138K, 46K, and 20K. Finally, the difference in migration rates of gp87 in SDS-polyacrylamide gels under reducing and nonreducing conditions suggests the existence of intramolecular disulfide bonds.

In vitro proliferation of lymphocytes from cyclophosphamide-pretreated mice immunized with antigen mixed with dimethyl dioctadecyl ammonium bromide.

A blastogenesis assay employing lymphocytes from cyclophosphamide-pretreated mice immunized with antigen mixed with the immunopotentiating compound dimethyl dioctadecyl ammonium bromide is described. The model antigen used for determining the assay parameters was inactivated purified measles virus. The optimal time for removal of immunologically primed T cells was 7 days after immunization of mice pretreated 2 days previously with 200 mg of cyclophosphamide/kg. The peak lymphoproliferative response was found to occur after 3-5 days in culture, depending on the concentration of antigen used. Although fetal bovine serum and syngeneic mouse serum each worked well as a medium supplement, significantly higher specific and lower non-specific lymphoproliferation were obtained when the mouse serum was used. Most of the lymphocytes responding to antigen were of the Ly 1.2 phenotype. Specificity of the blastogenic response was shown by a lack of cross-reactivity among measles virus, herpes simplex virus type 1 and vesicular stomatitis virus antigens. This approach to a mouse blastogenesis assay involves an easy way to induce strong T cell priming in mice, while still providing an assay which has an ideal combination of low non-specific and high antigen-specific responses.

Epitope mapping of the major Epstein-Barr virus outer envelope glycoprotein gp350/220.

To understand the complete immunochemical structure of the Epstein-Barr virus (EBV) major membrane glycoprotein gp350/220, monoclonal antibodies (MAbs) reacting with this important viral antigen were isolated. Through competitive inhibition binding studies, it was determined that a group of 18 IgG MAbs recognized seven distinct epitopes on the gp350/220 molecule. Eight of these MAbs fell into a single epitope group with four of those MAbs, as well as a single MAb from another epitope group, being capable of neutralizing EBV strain B95-8 transformation of umbilical cord lymphocytes.

Evidence that avian tumour virus-immune chicken sera recognize only viral structural antigens on the surface of avian tumour virus-infected cells.

The specificity of the humoral response of chickens to avian tumour viruses (ATV) was investigated by reacting ATV-immune sera with Triton X-100 extracts of uninfected, infected and transformed chicken embryo fibroblasts. Analysis of these immune reactions by polyacrylamide gel electrophoresis revealed that avian leukosis virus-challenged and Rous sarcoma virus-challenged chickens recognized only two major cell surface antigens of 100000 and 29000 mol. wt. which were present on transformed and non-transformed virus-producing cells. No labelled antigens were precipitated from uninfected cells or transformed cells producing the envelope-defective mutant RSV(-). The antigens were shown to be related to the major envelope glycoproteins of the virus and to contain group-specific determinants common to ATV subgroups B and C. No group-specific determinants common to ATV subgroups A and B or subgroups A and C were detected. Chickens were found to have a strong antibody response to the 100000 and 29000 mol. wt. proteins prior to and during tumour rejection, even in the absence of neutralizing antibody to the challenge virus. No tumour-specific surface antigen distinct from the virion structural antigens was detected by any of the immune chicken sera on any of the transformed cells tested.

Purification and biologic characterization of a major Epstein Barr virus-induced membrane glycoprotein.

The Epstein Barr virus membrane antigen (MA) complex is composed of three major high m.w. glycoproteins designated gp 300/350, gp 200/250, and gp 85/90. In the experiments reported in this paper, the gp 300/350 glycoprotein was purified from MA-positive extracts prepared from the B-95-8 cell line. This was accomplished by tandem combination of ion-exchange and lectin chromatography. It was routinely possible to purify this glycoprotein 400- 600-fold by this procedure. Sera from animals immunized with gp 300/350 purified by this approach neutralized EBV infectivity and mediated antibody-dependent cellular cytotoxicity (ADCC) demonstrating that both of these important antigenic determinants were expressed on this glycoprotein. The presence of an ADCC determinant on this molecule was further substantiated by the finding that monoclonal antibody to gp 300/350 blocked ADCC mediated by a human ADCC antibody-positive reference serum.

Identification of Epstein-Barr virus strain differences with monoclonal antibody to a membrane glycoprotein.

Two monoclonal antibodies directed against Epstein--Barr virus (EBV)-induced membrane antigens (MA) were isolated in this study. On of the monoclonal antibodies, designated 2F5.6, was an IgG2 which, as detected by membrane and fixed cell immunofluorescence, reacted with MA-positive lymphoblastoid cell lines that produced transforming EBV but not with the MA-positive P3HR-1 cell line that produced the lytic, nontransforming strain of this virus. This antibody precipitated the Mr 320,000/350,000 glycoprotein from B-95 virus infected cultures and the Mr 300,000 and 220,000/250,000 glycoproteins from Raji cells superinfected with P3HR-1 virus but did not precipitate any of these EBV-specific glycoproteins from the P3HR-1 cell line. In contrast, the second monoclonal antibody, IgM designated B10.3, reacted with all virus-producing cell lines including the P3HR-1 cell line. The identity of the glycoprotein that serves as the target for this antibody is still unknown. Neither antibody had neutralizing activity against the B-95 or P3HR-1 strain of EBV. These results indicated that the 2F5.6 monoclonal antibody was directed against an antigenic determinant on the major membrane glycoprotein which is common to transforming strains of EBV but absent from the lytic P3HR-1 stain whereas the B10.3 monoclonal antibody was directed against a group-specific EBV-induced membrane determinant.

IgA antibody, antibody-dependent cellular cytotoxicity and prognosis in patients with nasopharyngeal carcinoma.

Immunoglobulin fractions containing antibodies to the Epstein-Barr virus (EBV) were isolated from different sera and tested in the antibody-dependent cellular cytotoxicity (ADCC) assay. All cytotoxic activity resided in the IgG fraction. IgA antibodies were not cytotoxic in this assay against cells expressing the EBV-induced membrane antigen complex. However, IgA antibodies were able to block the IgG-mediated ADCC reaction, indicating that the IgA and IgG antibodies recognized the same EVB-specific antigenic determinants. This was supported by results from radioimmune precipitation experiments. Our findings suggest that low ADCC titers previously identified in the sera of patients with nasopharyngeal carcinoma (NPC) who had a poor prognosis could be due to the blocking activity of IgA antibodies. The results further suggest that IgA antibodies are detrimental to the patient with this disease, if one assumes that ADCC functions in vivo in immunity to this tumor

Purification and biochemical characterization of an inhibitor of DNA synthesis produced by an Epstein-Barr virus-transformed B cell line.

An inhibitory factor, which has been shown to suppress the uptake of 125I-iododeoxyuridine by both lymphoid and nonlymphoid cells, was isolated from the supernatant of an Epstein-Barr virus- (EBV) transformed B cell line (1605L) established from a cotton-topped marmoset. Purification of the inhibitor, which was produced in serum-free medium by crowded cultures of the 1605L cells, was achieved by DEAE-cellulose chromatography followed by preparative polyacrylamide gel electrophoresis. The apparent m.w. of the 1605L factor was determined to be 65,000 to 70,000 by SDS-polyacrylamide gel electrophoresis. The inhibitor was sensitive to digestion by trypsin and chymotrypsin but not RNase or DNase, indicating that it was protein in nature. Exposure of the 1605L factor to 56 degrees C for 1/2 hr or pH 2 for 48 hr at 4 degrees C destroyed its inhibitory activity. The biochemical characteristics and activity of the 1605L inhibitor distinguish it from Type I interferon and several other soluble immunologic mediators known to be produced by lymphoid cell lines.

Immunoglobulin A antibody to Epstein-Barr viral antigens and prognosis in nasopharyngeal carcinoma.

IgA immunoglobulin fractions containing antibodies to Epstein-Barr virus (EBV)-induced membrane antigens were isolated from the sera of two patients with nasopharyngeal carcinoma (NPC), from one non-NPC patient, and from three persons with sera negative for IgA antibodies. IgA antibodies were not cytotoxic against cells expressing EBV-induced membrane antigens in the antibody-dependent cellular cytotoxicity (ADCC) assay. However, IgA antibodies blocked IgG-mediated ADCC, which indicated that these antibodies could serve as a blocking "factor" in patients with disease and, therefore, were potentially detrimental to the host.

Epstein-Barr virus-specific antibody-dependent cellular cytotoxicity in patients with Burkitt's lymphoma.

Coded sera from 54 patients with African Burkitt's lymphoma (BL) were titrated for antibodies against an Epstein-Barr virus (EBV)-induced membrane antigen in the antibody-dependent cellular cytotoxicity (ADCC) assay. The titers were then correlated with the progression of lymphoma growth following chemotherapy. In 74% of the patients with high ADCC titers (greater than 3,840), lymphomas showed partial or complete regression following therapy. In the medium-titered group (240-3,840), 36% of the lymphomas showed some response to therapy, while only 29% of the lymphomas in the low group (less than 240) responded to treatment. These preliminary results indicated that, as previously reported for patients with nasopharyngeal carcinoma, ADCC titers may be a prognotic value in patients with this EBV-associated disease. In an attempt to determine the identity of the ADCC antigen, some of these sera were examined for antibody to the four major MA components so far identified in the membrane of EBV-infected Raji cells. Sera with high ADCC titers in general contained antibody to the four major MA components, while low-titered sera usually contained antibody to three or less of these proteins. There were exceptions to this pattern, however, indicating that the ADCC antigen might differ from the four EBV-induced membrane components so far identified.

Epstein-Barr virus-induced membrane antigens: immunochemical characterization of Triton X-100 solubilized viral membrane antigens from EBV-superinfected Raji cells.

In an attempt to qualitatively identify the membrane antigen (MA) complex induced by Epstein-Barr virus (EBV) infection of lymphoblastoid cells, superinfected Raji cells were surface labelled with 125I by the lactoperoxidase method and solubilized with Triton X-100, then the 125I-labelled membrane proteins were precipitated by sera containing high antibody titers to MA. Analysis of these immune precipitates on sodium dodecyl sulfate polyacrylamide gel eletrophoresis identified four major EBV-specific membrane proteins with molecular weights (mol. wt) of 280,000, 250,000, 170,000 and 90,000. Sera from patients with Burkitt's lymphoma (BL), nasopharyngeal carcinoma (NPC) and infectious mononucleosis (IM) and from EBV-infected disease-free individuals showed differential patterns of reactivity to these antigens with some sera only recognizing three or less of the antigens. The results from invesigations with these sera also indicated that these major proteins were not related to EBV-induced viral capsid antigens (VCA) or the virus-associated early antigen (EA) complexes as defined by immunofluorescence. Metabolic labelling of EBV-infected Raji cells with [14C]glucosamine, followed by Triton X-100 solubilization and radioimmune precipitation, identified the 280,000, 250,000 and 90,000 components as glycoproteins. The lactoperoxidase-labelled 170,000 molecular weight component was not significantly glycosylated and, therefore, could not be categorized as a glycoprotein on the basis of this study. In addition, a glycoprotein with a mol. wt of 130,000 was identified by this approach which also appeared to be specified by EBV. The results from these investigations, therefore, indicated that the EBV-induced MA complex was composed of four major glycoproteins and one nonglycosylated high mol. wt protein.