Two coronaviruses isolated from central nervous system tissue of two multiple sclerosis patients.

Two coronaviruses were isolated from brain material obtained at autopsy from two multiple sclerosis patients. The viruses were neutralized by serum and spinal fluid from these patients. Although most of the population have antibody to these virus isolates, multiple sclerosis patients have slightly higher concentrations of serum antibody than controls. The results suggest that coronaviruses should be considered as one additional virus with a potential implication in the etiology of multiple sclerosis.

Microcytotoxicity assays of tumor immunity in patients with bronchogenic carcinoma correlated with clinical status.

Peripheral blood lymphocytes from patients with bronchogenic carcinoma were tested in microcytotoxicity assays against cultured bronchogenic cancer cells, other types of tumor cells, and skin fibroblasts. Lymphocytes from patients who were postresection with no clinical evidence of residual or recurrent tumor were more frequently toxic against bronchogenic carcinoma than were lymphocytes from normal donors or from patients with clinically evident disease. Lymphocytes from patients with minimal or no tumor were more frequently toxic against bronchogenic cancer than against skin fibroblasts. Serum samples from a few patients rendered lymphocytes toxic for bronchogenic cancer cells, but this serum activity could not be correlated with the patient's clinical status.

Effects of human lymphocytes on cultured normal and malignant cells.

With a modified microcytotoxicity assay, the effects of lymphocytes from normal volunteers and from patients with a variety of solid malignant tumors were tested in vitro against both normal and neoplastic target cells. The study was divided into three sequential time phases in each of which a different method of lymphocyte separation was used. Lymphocytes were separated by a Ludox-polyvinyl-pyrrolidone technique in the first phase, by nylon wool filtration in the second phase, and by a Ficoll-Hypaque technique in the third phase of the study. In the first two phases of the study, lymphocytes from both normal volunteers and cancer patients were frequently toxic on both tumor cells and fibroblasts. In the last phase of the study types of target cells. Cancer patient lymphocytes were more frequently toxic on tumor cells but no more frequently toxic on fibroblasts than were normal lymphocytes. The different results obtained in the last phase of the study cannot be attributed solely to the different method of lymphocyte separation, since other factors, such as better growth status of the target cells and greater facility in performing the assays, might also be responsible. Results of the last phase of the study raise the possibility that both specific and nospecific reactions may contribute to the toxicity observed in this laboratory with cancer patient lymphocytes tested against tumor cells. It has not yet been possible to reduce the level or frequency of the nonspecific reactions to such a degree that the microcytotoxicity assay can be used clinically to document clearly or to follow the putative tumor-specific immunity of individual cancer patients.

Effects of human sera on reactivity of lymphocytes in microcytotoxicity assays.

The effects of cancer patient and normal donor serum samples on the reactivity of patient and normal lymphocytes against both normal and malignant target cells were studied in microcytotoxicity assays. There were 17 of 140 cancer patient serum samples and 7 of 116 normal donor lymphocyte samples that selectively increased the growth of target cells in the presence of lymphocytes. This effect was most often noted with cancer patient serum against cultured tumor cells, but the effect was also noted against fibroblasts and with normal serum against both fibroblasts and tumor cells. Of 140 cancer-patient serum samples, 11 selectively decreased target cell survival in the presence of lymphocytes compared to medium and compared to other serum samples. In the absence of lymphocytes these serum samples were nontoxic. The effect was not observed with any of the normal serum samples studied. The lymphocyte-dependent serum toxicity appeared to be selectively directed against tumor target cells.

Pathogenesis of coronavirus SD in mice. I. Prominent demyelination in the absence of infectious virus production.

Following intracerebral inoculation of 3- to 4-week-old C57 B16/J mice with coronavirus SD, 23% exhibited neurologic signs within the first week. However, only 6% died. Within the first week after inoculation (AI), we noted a panencephalitis. Prominent demyelination detected in the spinal cord on day 6 continued through day 29 AI. Demyelinated lesions in the spinal cord were either subpial with few inflammatory cells except for macrophages or perivascular with prominent accumulation of lymphocytes, plasma cells, and macrophages. Beginning on day 6 AI, IgG was detected in the lesions. Although an infectious virus was detectable in the CNS only through day 12 AI, viral antigen expression continued through day 24. We concluded that coronavirus SD persists in a nonrecoverable form throughout the initial phase of demyelination, day 6 to day 24 AI.

Antigenic relationships of coronaviruses detectable by plaque neutralization, competitive enzyme linked immunoabsorbent assay, and immunoprecipitation.

The antigenic relationships of mouse coronaviruses JHM and A59, human viruses OC43 and 229E, and multiple sclerosis (MS) isolates SD and SK have been investigated by plaque neutralization, competitive enzyme linked immunoabsorbent assay, and immunoprecipitation. A59, SK, or SD plaques are neutralized by antiserum prepared against homologous as well as heterologous virus. Plaque neutralization also demonstrated weak reactivity between SD or SK and mouse virus JHM but no reactivity with human coronavirus 229E. An antiserum prepared against human virus OC43 neutralized viruses SD and SK but not mouse viruses A59 or JHM. In a competitive enzyme linked immunoabsorbent assay (cELISA) the binding of antiserum prepared against MS isolate SK to bound SK antigen was inhibited to a comparable degree using OC43, SD, or A59 viral antigens. Coronavirus 229E or uninfected cell antigens did not block the binding of anti-SK serum to bound antigen. However, a cELISA utilizing OC43 as bound antigen and competing an anti-OC43 serum suggests that virus OC43 may be more closely related to SK than A59. Specific viral polypeptides that share antigenic determinants have been identified by immunoprecipitation of S35 methionine labeled viral infected cell extracts. Polypeptides of similar molecular weight were precipitated from A59, SD, or SK infected cell extracts by SD, SK, OC43, or A59 antisera. Our data suggests that the mouse coronavirus A59, human coronavirus OC43, and MS isolates SD and SK contain antigenically related polypeptides of similar molecular weight.

Leukocyte adherence inhibition as measured by a radioisotopic technique for detection of cell-mediated tumor immunity.

It has been previously reported that cell-mediated tumor immunity may be detected by leukocyte adherence inhibition (LAI) as determined by a visual counting technique in the presence of soluble tumor antigen. In the present experiments leukocyte adherence was measured by a radioisotopic technique. Peripheral blood mononuclear cells from Wistar/Fu rats and lymph-node cells (LNC) from C3H/J mice were labelled with Technetium 99m (99mTc) and incubated with medium alone and with medium containing tumor antigens (high dilutions of dialyzed tumor homogenates) in the wells of plastic tissue-culture plates. After incubation, fluid and non-adherent cells were removed. The ratio of residual radioactivity in wells with antigen to that in wells without antigen was determined. Adherence of blood mononuclear cells from rats bearing syngeneic transplants of a polyoma-virus-induced tumor was inhibited by dialyzed homogenates of this tumor. Adherence of blood mononuclear cells from normal rats was not inhibited. Adherence of LNC from mice bearing syngeneic transplants of a 3-methylcholanthrene (3-MCA)-induced fibrosarcoma was inhibited by dialyzed homogenates of this tumor. Adherence of LNC from normal mice or from mice or from mice bearing a different 3-MCA-induced tumor was not inhibited. These results support the use of LAI as a simple test for cell-mediated tumor immunity. Measuring relative leukocyte adherence with the radioisotope 99mTc eliminates possible bias in counting and permits multiple replicate determinations with a small number of leukocytes.

Coronavirus isolates SK and SD from multiple sclerosis patients are serologically related to murine coronaviruses A59 and JHM and human coronavirus OC43, but not to human coronavirus 229E.

Two coronaviruses (SK and SD), isolated from fresh autopsy brain tissue from two multiple sclerosis patients, were compared with known human and murine coronaviruses. In plaque neutralization assays, antisera prepared against multiple sclerosis isolates SK and SD demonstrated significant cross-reactivity to each other and to murine coronavirus A59, weak cross-reactivity to murine coronavirus JHM, but no cross-reactivity to the human coronavirus 229E. Antiserum to SK or SD failed to inhibit hemagglutination of chicken erythrocytes by the human coronavirus OC43. However, OC43 antiserum neutralized both SD and SK. Specific coronavirus polypeptides were identified and compared by immunoprecipitation and polyacrylamide gel electrophoresis. Infected and mock-infected 17Cl-1 cells were pretreated with actinomycin D and labeled with [35S]methionine. Polypeptides in Nonidet P-40 cytoplasmic extracts were immunoprecipitated with homologous and heterologous antisera. Identical polypeptides were precipitated from A59-, SD-, or SK-infected cell extracts by SD, SK, OC43, or A59 antisera. The polypeptides of human virus 229E were antigenically distinct, with the exception of weak recognition of a polypeptide of 50,000 molecular weight. We conclude that the two multiple sclerosis virus isolates SK and SD are closely related serologically to the murine coronavirus A59 and the human coronavirus OC43.