Abnormalities of in vitro lymphocyte responses during rubella virus infections.

In vitro rubella virus infections of lymphocytes from normal adult humans impaired their responsiveness to phytohemagglutinin (PHA) stimulations; a situation which seemed analogous to the PHA unresponsiveness of peripheral lymphocytes from babies with the congenital rubella syndrome. Such in vitro viral infection of normal cells also decreased the synthesis of normal nucleic acids and structural proteins, and abrogated the enhanced DNA synthesis induced by pokeweed and specific antigen stimulations. Furthermore, it was shown that live rubella virus, but not ultraviolet-irradiated virus, was necessary for the impaired mitogenic responses of normal leukocytes. These observations are interpreted to favor the view that the virus achieves its inhibitory effect on the action of mitogens by interference either directly or indirectly at an intracellular site. Such an action could reduce the functional potential of lymphocytes and impair their effectiveness as immunologically competent cells or as effectors in immunologic reactions.

Effect of amantadine hydrochloride on the response of human lymphocytes to phytohemagglutinin.

Amantadine hydrochloride (Symmetrel), which is an antiviral agent marketed for the prevention of infection by influenza virus, inhibits the mitogenic response of human lymphocytes stimulated with phytohemagglutinin. The concentrations which effectively inhibited the response to phytohemagglutinin were similar to those which maximally inhibit virus replication. The drug inhibited the mitogenic response without affecting the ability of phytohemagglutinin to agglutinate leukocytes. The data suggest that phytohemagglutinin, amantadine, and certain lipid-containing RNA viruses take part in cell-membrane interactions of common nature.

Viral inhibition of lymphocyte response to phytohemagglutinin.

The response to phytohemagglutinin by lymphocytes from eight of fourteen patients with congenital rubella was inhibited, whereas that of lymphocytes from patients with other diseases was not. The response of normal lymphocytes infected in vitro was also inhibited. The results suggest that early association of lymphocytes with virus inhibits the function of the cell and contributes to persistent carrying of virus in congenital rubella. This phenomenon may be a means of detecting viruses not now recognizable by routine methods of tissue culture.

Characterization of human maligant melanoma cell lines. I. Morphology and growth characteristics in culture.

Seven human malignant melanoma lines were maintained in vitro for various periods of time. One line, established in this laboratory from a metastatic solid tumor by repeated treatment of the primary outgrowth with 0.02 percent EDTA, allowed a continuous culture of melanoma cells free of fibroblasts. By light microscopy, cells in each line could be classified into one of three morphologic types: elongated dendritic, cuboidal, or triangular dendritic. Four of the seven lines exhibited various degrees of pigmentation. The growth pattern was determined by plating efficiency and saturation density for each line. Cytogenetic analysis with the fluorescent banding technique revealed only human chromosomes with gross aneuploidy. Major marker chromosomes specific for each line were identified. None of the parameters studied showed any correlation or interdependence with one another, except for an association of elongated dendritic morphology with poor plating efficiency and low saturation density.

Detection of new cross-reacting carcinoembryonic antigen(s) on cultured tumor cells by mixed hemadsorption assay.

Antisera highly specific for carcinoembryonic antigen (CEA) from New Zealand White rabbits and a goat reacted strongly in antibody binding tests with cultured tumor cell lines, irrespective of the ability of the cell lines to produce CEA. The most reactive were colon carcinoma and melanoma cell lines, the former known to produce CEA and the latter not associated with CEA production. The reactivity was not diminished by absorption with perchloric acid extracts of normal lung or spleen, whereas absoprtion with purified CEA preparations abolished the reactivity. Quantitative absorption studies indicated that reactivity against CEA-producing cell lines could be totally removed by absorption with other CEA-producing lines but not with melanoma cell lines. Reactivity against melanoma cell lines could be completely removed by colon carcinoma cells as well as by melanoma cells. Antisera raised against purified CEA, after absorption with extracts of normal lung, still contained two populations of antibodies, one that binds a newly described antigen cross-reacting with CEA which is present on melanoma cells.

Enhanced expression of melanoma-associated antigens and beta 2-microglobulin on cultured human melanoma cells by interferon.

The effect of human leukocyte interferon (IFN) on the in vitro growth and expression of melanoma-associated antigens (MAA). beta 2-microglobulin (beta 2m) and HLA-DR antigen on cultured human melanoma cells was studied. Exposure of melanoma cells to IFN for 64 hours resulted in a dose-dependent inhibition of growth with 46% reduction in cell number at 10(3) U IFN/ml and 74% reduction at 10(5) U/ml. Quantitative absorption experiments in the mixed hemadsorption assay determined that the expression of MAA and beta 2m on treated cells was enhanced at 10(2)-10(5) U IFN/ml, twofold to fivefold for MAA and fivefold to twelvefold for beta 2m. No change was seen in HLA-DR antigen expression. The IFN-induced enhancement of MAA and beta 2m could be detected as early as after 16 hours and a maximum expression was reached at 96 hours after IFN exposure. The IFN-induced enhancement of MAA and beta 2m on melanoma cells was reversible. Studies with melanoma cells grown in stationary phase and serum-deprived conditions indicated that IFN-induced augmentation of MAA and beta 2m did not require cell proliferation. The data suggest that the effect of IFN on antigen expression is independent of its effect on cell growth. Further studies are needed to fully elucidate the mechanism.

Sensitivity of human carcinoma cell lines to lysis by blood natural killer cells correlating with surface expression of carcinoembryonic antigen.

The HCT-8R clone of the HCT-8 human colon tumor line, which expresses increased quantities of carcinoembryonic antigen (CEA) on its surface, was discovered to have an enhanced susceptibility to lysis by natural killer (NK) cells in human peripheral blood. This increase in susceptibility to lysis by peripheral blood mononuclear cells was not explained by stimulation of interferon release by HCT-8R cells but rather was found to be attributable to an increased susceptibility of HCT-8R cells to lysis by those NK cells that bind to sheep erythrocytes (E-RFC). Cold target competition experiments and single-cell assay for cytotoxic cells suggested that the presence of surface CEA did not increase lysis of HCT-8R by facilitating "recognition" by E-RFC-type cytotoxic cells but by rendering HCT-8R cells more susceptible to the lytic mechanism of NK cells. The magnitude of expression of surface CEA by a variety of human carcinoma cell lines with a few exceptions and subclones of HCT-8 also correlated with increased susceptibility to lysis by blood mononuclear cells. The possible clinical significance of these findings was discussed.

Monoclonal antibody recognizing human melanoma-carcinoma cross-reacting oncofetal antigen epitopically associated with carcinoembryonic antigen.

By fusion of mouse NS1 myeloma cells with splenocytes from a BALB/c mouse immunized with human melanoma cells, an IgG1 monoclonal antibody, designated as 140.72, was produced. By the mixed hemadsorption antibody binding assay, 140.72 was shown to react with 17 of 20 melanoma cell lines and with 5 of 14 carcinoma cell lines. This antibody also reacted with 3 of 3 normal melanocyte cultures in much lower titers. It did not react with any of 35 other normal and malignant lines, including neuroblastoma, glioblastoma, sarcoma, teratoma, fibroblast, and lymphoid cell lines. Absorption with fresh melanoma and carcinoma homogenates confirmed the results of direct tests. Fetal reactivity of antibody 140.72 was determined by positive absorption with 10 of 11 tissue homogenates derived from different fetuses of 10-16 weeks' gestation. The reactivity of this antibody was completely removed by absorption with a highly purified preparation of carcinoembryonic antigen (CEA) derived from a colon carcinoma. The antigenic activity was detected in the culture medium of reactive cell lines. Immunoprecipitation analyses of melanoma and carcinoma cells indicated that the antigenic determinant recognized by antibody 140.72 is on a glycoprotein with an apparent molecular weight of 95,000-150,000 common to both serologically reactive cell types. Additionally, a 200,000-molecular-weight glycoprotein corresponding to the CEA molecule was detected only on the reactive carcinoma cells. These data confirmed previous findings obtained with polyclonal anti-CEA antisera for the existence of shared CEA-related antigenic determinants on human carcinomas and melanomas and provided additional molecular characterization of these glycoproteins. Further characterization of the molecules bearing the antigenic determinant recognized by antibody 140.72 should be performed with a view to exploring its potential in the immunodiagnosis and immunotherapy of patients with melanoma.

Common and individually specific chromosomal characteristics of cultured human melanoma.

Since individual chromosomes can be accurately identified by new banding techniques, atebrin fluorescence was used for chromosome analysis in six cell lines and two primary outgrowths derived from human malignant melanoma. Gross aneuploidy was seen in all specimens, but each culture contained at least 1 distinctive marker chromosome specific for that cell line in 87 to 100% of metaphases. One of the primary explants contained a marker that was demonstrable in fresh tissue and persisted through 2 weeks of culture. The same marker was found in all metaphases from 2 different metastases, but skin fibroblasts from the same patient had a normal chromosome complement. No common marker for human melanoma was found, but in 6 of the 8 cultures the most frequently found marker was formed by a brightly banded chromatid addition. Relative polysomy for Chromosome 7 was found in 7 of the 8 cultures and, for Chromosome 22, in 8 of the 8 cultures. The frequency of polysomy of Chromosomes 7 and 22 was significant at the 5% level.

Characterization of human malignant melanoma cell lines. IV. Cytologic and histochemical characteristics.

The cytologic and histochemical characteristics of seven established human melanoma cell lines were studied using monolayer culture and cytocentrifuge perparations. The morphology of the cells is different in the two preparations. There is a strong resemblance between the Cytocentrifuge preparations and the histological tissue sections and cytologic smears that are used in routine diagnostic work.