Biomedical subjects
Publications and source records attributed to R Szigeti.
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Leukocytes from EBV-seropositive but not seronegative healthy donors responded with significant migration inhibition to the 48K subcomponent of the Epstein-Barr virus determined nuclear antigen (EBNA), known to carry the virally determined antigenic specificity. A concentration of 10 micrograms/ml was still effective while 5 micrograms/ml had no detectable effect. EBNA-associated cellular 53K protein had no effect by itself, but it potentiated the effect of 48K, even if the latter was added at the subliminal concentration of 5 micrograms/ml. The related 53K protein, isolated from EBV-negative human lymphoma cells, was also effective, whereas the corresponding murine-tumor-associated 53K had no potentiating effect. Immunization of mice with an extract of DNA-binding proteins from EBV-carrying Raji cells, known to contain both 48K and 53K, induced a significant macrophage migration inhibition response, to both human 48K and 53K. Murine 53K was ineffective, however. Human but not murine 53K increased the migration inhibitory activity of subliminal concentrations of 48K in the murine macrophage system as well. These findings suggest that human but not murine 53K may reconstitute with 48K (EBNA) to form a highly immunogenic complex.
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Production of leukocyte migration inhibitory factor (LIF) by fresh and cryopreserved lymphocytes from the same donors was detected by indirect leukocyte migration inhibition (LMI) assay. The same results were obtained when fresh and frozen lymphocytes were tested in parallel. This indicates that cryopreservation does not impair the ability of lymphocytes to produce LIF.
Three males with the X-linked lymphoproliferative syndrome (XLP) with hypo- or agammaglobulinemia following Epstein-Barr virus (EBV) infection and two males with the chronic mononucleosis syndrome were investigated for immune responses to EBV-determined antigens. Males with XLP showed profound cellular immune defects. Markedly diminished responses of natural killer cell and interferon-activated killer cell activities and impaired leukocyte migration inhibition responses to phytohemagglutinin were determined in patients with XLP. The two patients with chronic mononucleosis showed less severe defects. All patients showed partial or complete impairment of their EBV-specific immune responses as measured by leukocyte migration inhibition. EBV-specific antibodies were markedly diminished against EBV-associated nuclear antigen, early antigen, and viral capsid antigen in males with XLP. In contrast, patients with chronic mononucleosis had elevated antibodies to most EBV-specific antigens. Individuals with life-threatening EBV-induced lymphoproliferative disorders may exhibit multiple defective immune mechanisms against the virus.
We have previously shown that the leukocytes of healthy EBV-seropositive (but not seronegative) donors respond with migration inhibition (LMI) when confronted with extracts of EBV-carrying (but not EBV negative) cells. In the present study, we have examined whether this EBV-specific LMI response is capable of detecting a membrane antigen on the surface of EBV-carrying virus nonproducer cells. Crude membranes from EBV-genome carrying and EBV-negative cell lines were used as antigen. Contamination with the EBV-determined nuclear antigen (EBNA) was ruled out. Membranes from EBV-genome carrying nonproducer cells inhibited the migration of leukocytes from healthy seropositive donors, whereas membranes from EBV-negative lines had no such effect. Seronegative donors did not show any LMI. The clear difference between the EBV-negative Ramos line and its EBV-converted sublines was particularly conclusive in showing that the membrane component is determined or induced by the viral genome.
After Epstein-Barr virus (EBV) infection in vivo, B-cells with latent virus infection persist indefinitely through life. These cells grow in vitro on explanation and can be established as immortal B-cell lines. To reconcile the unlimited growth potential in vitro with the maintenance of a low proportion of B-cells infected by EBV in vivo, a strict in vivo control mechanism has to be postulated. Certain aspects of this control are apparent when the primary infection is followed by infectious mononucleosis. This is characterized by lymphocytosis and the presence of activated T-cells. The T-cell proliferation is probably the manifestation of the immune response against EBV antigens. However, the reaction of T-cells upon encounter of B-blasts is also likely to contribute to the events. At present, it is difficult to detect an EBV-specific component in the action of the T-cells in the acute phase of mononucleosis exerted on B-cells. However, for the clinical course of the disease the activation of T-cells is important. The activated T-cells may control and also eliminate the B-cells infected by EBV. In addition to the immunity which develops during the disease, th immunoregulatory mechanism is likely to have a role in the inhibition of B-cell proliferation.
Two patients with Hodgkin's disease in remission and one chronic lymphatic leukemia patient with extraordinarily high anti-Epstein-Barr virus (EBV) (viral capsid antigen) antibody titers (greater than 10,000) were selected to study a spectrum of cell-mediated immune responses, including natural killer, interferon-boosted killer, antibody-dependent lymphocytotoxicity, and T-cell-mediated reactions. The purpose was to compare these reactions in patients with immunosuppression and a high EBV load who can hold their EBV-carrying cells under control with the corresponding reactions in patients with EBV-carrying lymphoproliferative disease. In contrast to the latter group, the three patients of the present study showed a less profound and less general suppression of the immune responses. Multiple effector mechanisms probably safeguard against the proliferation of EBV-transformed B-cells. Clinically manifest EBV-carrying lymphoproliferative disease occurs only in very severe immunodeficiencies effecting multiple effectors.
A six-month-old male infant, whose elder brother had died of progressive vaccinia due to combined immunodeficiency, contracted varicella-zoster infection, and died of disseminated varicella ten days after onset of the disease. As with his elder brother, the blood levels of immunoglobulins were normal, and specific varicella antibodies appeared in his serum, although in low concentrations. His T-lymphocytes, although their number was subnormal, could be stimulated with phytohaemagglutinin, and production of migration inhibitory factor could also be demonstrated. The necropsy confirmed thymic dysplasia. In addition to histological changes, severe combined immunodeficiency and foci of malignant lymphoma were present. On this basis the disease was classified, according to the WHO recommendations, as severe combined immunodeficiency with B lymphocytes, complicated by malignant lymphoma.
Although first recognized by its effect on virus-cell interactions, interferon (IFN) has a variety of other effects. It can affect cell proliferation, modify the immune response at several levels, enhance the cytotoxic action of lymphocytes, suppress antibody formation and inhibit the development of delayed-type hypersensitivity (DTH) reactions. Therefore we have now tested the effect of interferon on leukocyte migration inhibition (LMI), regarded as the counterpart in vitro of DTH in humans. We have found that IFN suppresses both mitogen-and antigen-induced LMI, acting directly on the granulocytes but also affecting the lymphokine production of the lymphocytes.
Interferon-alpha 1 from Escherichia coli transformed with a hybrid plasmid containing a human leukocyte complementary DNA insert, induces resistance to virus in appropriate target cells. It also shares the following properties with natural leukocyte interferon (IFN). (i) It enhances natural killing activity of human lymphocytes, (ii) it enhances antibody-dependent cell-mediated cytotoxicity, (iii) it suppresses antigen- and mitogen-induced leukocyte migration inhibition, (iv) it inhibits growth of IFN-sensitive Burkitt lymphoma cells. Since these activities are exhibited by a cloned protein species, they are due to IFN itself and not to other human proteins.
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Leukocyte migration inhibition of seronegative and seropositive healthy children and that of patients with infectious mononucleosis was studied in the presence of EBV-negative and -positive cell extracts. Seronegative children showed no alteration of the leukocyte migration in the presence of the extracts. EBV-positive cell extracts caused significant migration inhibition in the group of seropositive healthy children and of IM patients. The effect was specific, since the EBV-negative cell extract had no inhibitory effect. In the acute phase of infectious mononucleosis a depressed response was observed, while after recovery the same migration inhibition could be detected. The possible cause of the depressed cell-mediated immune response in the acute phase of the disease is discussed.
Phytohaemagglutinin-induced leukocyte migration inhibition was studied in 72 children and compared with phytohaemagglutinin skin testing. The methods were used for screening the cell-mediated immune response. The in vitro method, which requires only 10-15% of the number of migrating cells as compared with other migration techniques, proved to be more sensitive, but gave some false-positive results when compared with other immunological parameters. Nevertheless, application of the microdroplet leukocyte migration is advantageous, used either simultaneously with the skin test, or as a single screening method in prematures, mature newborns and young infants, where application and/or evaluation of the skin tests is difficult.
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