Robert Szigeti

Virologic, immunologic, and clinical observations on a patient during the incubation, acute, and convalescent phases of infectious mononucleosis.

One patient with infectious mononucleosis (IM) was studied from the probable time of Epstein-Barr virus (EBV) infection (38 days before the onset of clinically overt disease), during the incubation and acute phases, until 6 months after clinical remission. Analysis of spontaneous outgrowth of EBV-carrying lymphoblastoid cells, by limiting dilution on feeder layer cultures, showed that virus containing B lymphocytes are already present early during the incubation period. Also low interferon serum levels were detected early after infection, and only before the onset of clinical disease. All other studied clinical laboratory and virus-associated variables were within normal range during the incubation phase, but changed to a pattern characteristic of IM in parallel to the clinical symptoms. During the acute disease EBV-associated nuclear antigen (EBNA)-positive cells could be directly detected among the lymphocytes, and antibodies to EBV antigens appeared. Lymphocytes stained by monoclonal antibodies, detecting Ia-like determinants (activated cells) and suppressor cells, increased dramatically, in parallel to a strong increase of functional suppressor cell activity, measured by inhibition of blastogenesis and PWM-induced immunoglobulin production. During the acute phase there was also a decrease of spontaneous cytotoxicity against the NK-sensitive cell line K562, while cytotoxicity (spontaneous) against an autologous EBV-positive lymphoblastoid cell line (LCL) was detected only during this phase. These reactions correlated to the presence of blasts, and the autologous reaction was exerted mainly by Fc-receptor-negative cells. Lymphokine production in response to EBV antigens was also initiated during the acute phase. During the convalescence period the serological and cellular immune parameters adjusted to the pattern of a normal EBV-seropositive person.

Characterization of the serological response in man to the latent membrane protein and the six nuclear antigens encoded by Epstein-Barr virus.

A total of 116 sera from healthy individuals and from patients with Burkitts lymphoma (BL), nasopharyngeal carcinoma (NPC) or rheumatoid arthritis (RA) were studied with respect to antibody responses to each of the seven known transformation-associated Epstein-Barr virus (EBV)-encoded antigens [latent membrane protein (LMP) and six nuclear proteins (EBNAs 1 to 6)]. The antibodies were detected using modified standard immunoblotting techniques. Antibodies to LMP were detected for the first time in sera from 6/27 (22%) healthy, EBV-immune individuals (seropositive for the viral capsid antigens). An increased incidence of anti-LMP antibodies was found in EBV-immune sera from patients with BL (17/24 positive; 71%), NPC (21/33; 64%), and RA (16/21; 76%). Antibodies to EBNA 1 were detected in all EBV-immune sera at a standard 1:20 dilution. Antibodies to the other EBNAs were detected in only a proportion of these sera (20 to 95%) at the same dilution. Only minor disease-associated differences in the incidence of these antibodies were observed, the most consistent being that RA sera had a higher incidence of antibodies to EBNAs 2 to 6 compared with healthy controls. Testing of the sera at a 1:100 dilution suggested that there were some disease-related differences in the titres of anti-EBNA antibodies. At this serum dilution, a reduced incidence of antibodies to EBNA 2 was seen in NPC (6/31) compared with RA (18/19) and healthy EBV-seropositives (16/26); antibodies to EBNA 3 were detected at an increased incidence in BL (8/15) and NPC (16/31) compared with control sera (7/26); antibodies to EBNA 4 were detected at increased incidence in BL (5/15) and RA (6/19) compared with control sera (1/26); and antibodies to EBNA 6 were detected at increased incidence in NPC (19/31) and RA (7/19) compared with control sera (3/26).

Effect of different Epstein-Barr virus-determined antigens (EBNA, EA, and VCA) on the leukocyte migration of healthy donors and patients with infectious mononucleosis and certain immunodeficiencies

Abstract Leukocytes from healthy Epstein-Barr virus (EBV)-seropositive (SP) subjects show significant migration (LMI) with EBNA (EBV-determined nuclear antigen)-containing extracts derived from nonproducer, EBV-genome-carrying cells, or presented in the form of partially purified EBNA-preparation. Additional EBV antigens, EA and VCA, presented in the form of induced cell extracts do not add any measurable increase to their response. Leukocytes from acute infectious mononucleosis (IM) patients, on the other hand, do not respond to EBNA, but they show a good response to induced EA- or EA and VCA-containing cell extracts. This is in accordance with known differences in EBV-antibody pattern in healthy SPs as contrasted to acute IM patients. Leukocytes from chronic mononucleosis patients resemble those from acute IM patients in their preferential EA/VCA and deficient EBNA response in the LMI test. Cells from four X-linked lymphoproliferative disease (XLP) patients showed complete absence of reactivity to PHA and to EBV antigens in the LMI test, showing a general defect in cell-mediated immune responses. Leukocytes from a group of patients with lymphoproliferative malignancies in remission and elevated EBV-antibody titers showed a variety of responses, resembling normal SP donors, or IM patients, or showing a complete unresponsiveness in the LMI test.

Leukocyte migration inhibition studies with Epstein-Barr virus (EBV) determined nuclear antigen (EBNA) in relation to the EBV-carrier status of the donor

Abstract Cell extracts from EBV-genome-carrying cell lines inhibit the migration of leukocytes from EBV-positive but not seronegative healthy donors. In the present study extracts from EBV-negative lines and their own in vitro EBV-converted sublines were used to induce migration inhibition with leukocytes from seronegative and seropositive individuals. A clear difference was found between the extracts from EBV-negative and positive cell lines. Significant migration inhibition could be obtained with antigen(s) associated with the virus nonproducer state. Since EBNA is known to be expressed by all nonproducer EBV-genome-carrying cells, we have compared the effect of partially purified EBNA and correspondingly prepared mock-EBNA on the leukocyte migration. Purified EBNA inhibited the leukocyte migration of EBV seropositives, whereas mock-EBNA had no such effect.

Epstein-Barr virus (EBV)-specific cell-mediated and humoral immune responses in ataxia-telangectasia patients

As a part of studies on cell-mediated immune (CMI) responses of immunocompromised, Epstein-Barr virus (EBV)-infected patients who can or cannot restrict the proliferation of EBV-transformed B cells, we have studied 16 Turkish patients with ataxia-telangectasia (AT). Fifteen were EBV seropositive; one was seronegative. Among the seropositives, eight had no or only low anti-EBV-determined nuclear antigen (EBNA) antibody titers, while seven had normal anti-EBNA levels. EBV-seropositive and -seronegative healthy Turkish children were used as controls. We have particularly asked the question whether low EBNA antibody titers can be correlated with the level of EBV-specific and -nonspecific cell-mediated immunity. Non-EBV-specific tests included cell count, phenotypical characterization with monoclonal antibodies, assessment of natural killer (NK)-cell activity, and ability to suppress mitogen-induced immunoglobulin production. Two EBV-specific CMI tests were used: outgrowth inhibition (OI) and leukocyte migration inhibition (LMI). The majority of the patients of the low-EBNA antibody group was IgA deficient and had high levels of α-fetoprotein (a-FP). Cells reacting with OKT8 monoclonal antibody predominated in both AT patient groups. In contrast, the suppressor activity was present in only a few patients and NK and interferon-activated killing (IAK) activities were normal. EBV-specific cell-mediated responses were defective in seven of eight patients in the low-anti-EBNA group and five of seven patients in the group with normal anti-EBNA titers. It is concluded that AT patients are often defective in their EBV-specific cell-mediated immune responses and with regard to their EBNA antibody levels. These defects are associated with a predominance of T cells reacting with OKT8 monoclonal antibody.

Production of leukocyte migration inhibitory factor (LIF) in human lymphocyte subsets exposed to polyclonal activators

Lymphocyte subsets separated on the basis of nylon-wool adherence and E and EA rosetting, and characterized for the presence of esterase-positive phagocytic cells were investigated for production of leukocyte migration inhibitory factor (LIF) in response to polyclonal T- and B-cell activators, PHA, ConA, PWM, and Epstein-Barr virus (EBV). In the nylon-passed population only the high avidity E+EA+ cells responded to ConA, PHA-induced LIF production in all E-rosetting subsets. The nylon-adherent E+ subset, which contains activated T cells, produced LIF spontaneously. B cells produced LIF when exposed to PWM or uv-inactivated EBV. In accordance with the known T-cell dependence of PWM activation, LIF was detected only in supernatants of reconstituted populations containing both B and T cells. In contrast, uv-inactivated EBV, devoid of transforming potential, elicited LIF production in the pure B-cell population. LIF production in response to polyclonal activators seemed to be independent of accessory cells since reconstitution with autologous macrophages or semipurified monokine, high-molecular-weight Interleukin 1 (IL-1), did not alter the results.

Use of cryopreserved lymphocytes for the indirect leukocyte migration inhibition assay.

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.

Application of migration inhibition techniques in tumor immunology.

Publisher Summary Macrophage migration inhibition (MMI) and leukocyte migration inhibition (LMI) phenomena are in vitro correlates of delayed hypersensitivity, and migration inhibition assays are used to evaluate cellular immunity to tissue, tumor, and virus antigens. Any nontoxic immunogenic material can be applied as antigen in these assays at proper concentrations, which must be determined in preliminary, pilot experiments. Technically these assays are relatively simple and can be evaluated after 18-96 hrs, depending on whether the direct or indirect technique is applied. The advantage of these assays can be used in allogeneic combinations of cells, because during the short incubation period no apparent sensitization and reactivity to histocompatibility antigens can be found. Another advantage is that tests can be performed with cell extracts and cell fractions. The vast majority of animal and human tumors studied with MMI and LMI appeared to contain tumor-determined (TAA, TSTA) antigen, usually not present in normal adult tissue. The tumor-determined antigens are always immunogenic in autochthonous/autologous and usually in allogeneic tumor-bearing animal/patients.

Epstein-Barr virus (EBV) antigen-specific leukocyte migration inhibition (LMI) in infectious mononucleosis (IM): I. Kinetics and response to a membrane protein on EBV-transformed cells

Cell-mediated immune response of mononucleosis (IM) patients to Epstein-Barr virus (EBV)-determined antigens was measured by the leukocyte migration inhibition (LMI) assay. Patients in the acute phase of the disease failed to respond to partially purified nuclear antigen, EBNA, or to cell extracts that contained EBNA as the predominant EBV antigen. They showed a strong specific response to cell extracts enriched in early antigen (EA) and virus capsid antigen (VCA). The LMI response to EBNA appeared in convalescence in parallel with EBNA-specific antibodies, slightly later in children than in adults. Membrane fractions of EBV-carrying, virus nonproducer Raji cells induced an EBV-specific LMI at approximately the same time. A bacterial fusion protein containing the hydrophilic part of the virus-coded membrane antigen of latently EBV-infected cells also induced an EBV-specific response that parallelled the LMI reaction elicited by the Raji membrane fraction. This is in line with our previous finding (D. Sulitzeanu et al., J. Virol. 58, 230, 1986) that this fusion protein shares an epitope with Raji cell membranes.

EBNA-specific LIF production of human lymphocyte subsets

Using the indirect leukocyte migration inhibition technique T cells have been identified as being responsible for Epstein-Barr virus nuclear antigen-induced specific leukocyte migration inhibitory factor production. The response was dependent on the presence of macrophages or their product, T-lymphocyte activating factor.