Grace B. Cannon

Cytostasis of tumor cell lines by human granulocytes

Abstract Purified peripheral blood granulocytes from normal adult donors were tested for cytolytic and cytostatic activity against a variety of tumor-derived, virus-transformed, and normal cell lines. Altogether, 45 donors and 16 cell lines were tested. Although granulocytes mediated antibody-dependent cell-mediated cytolysis, no spontaneous cytolysis, as measured by chromium-51 ( 51 Cr) or [ 3 H]thymidine ([ 3 H]TdR) release could be detected in assays performed for up to 12 hr, even at an effector:target (E:T) cell ratio of 100:1. In contrast, granulocytes exhibited substantial growth-inhibitory activity (GIA) against most target cells, as measured by uptake of [ 3 H]TdR by the target cells. These results were confirmed by visual counting of target cells. The degree of cytostasis was dependent on the E:T ratio, with a plateau of 80–95% inhibition usually reached at a ratio of 40:1. Inhibition of growth of adherent tumor target cells was accompanied by cell detachment, with both effects apparent by 5 hr and reaching a peak after 15 hr of incubation. With nonadherent targets, the onset and the peak of cytostasis were delayed, being observed after 8 and 24 hr, respectively. Growth of target cells remained inhibited for up to 4 days of culture. A wide variety of target cells were sensitive to granulocyte-mediated cytostasis, including tumor-derived human and mouse cell lines, lymphoblastoid cell lines from normal donors, and embryo fibroblasts. Normal human fibroblasts were inhibited only at high E:T ratios (40:1). PHA-induced lymphoblasts were the only target cells tested that were completely resistant to the cytostatic effects of granulocytes and in fact, their growth was slightly stimulated. There appeared to be two somewhat different mechanisms of growth inhibition by granulocytes, which varied with the target cell. Trypsinization of granulocytes markedly reduced their reactivity against adherent target cells but had little effect on GIA against suspension target cells. Also, the activity against F-265, but not against other target cells, was almost completely abrogated in the presence of catalase, suggesting an important role of hydrogen peroxide in one mechanism of granulocytemediated cytostasis.

Carcinoma of the lung: Immunotherapy with intradermal BCG and allogeneic tumor cells

Abstract After lobectomy or pneumonectomy 45 patients with stage I and 6 with stage II non-small cell carcinoma of the lung were randomized to receive: no further therapy; intradermal BCG; or BCG plus allogeneic irradiated tumor cells intra-dermally twice monthly for 24 months. Patients were observed for 2–51 months at this writing. The homogeneity of the disease free interval (DFI) (i.e., differences in the pattern of relapses) was tested for the three groups. Analysis indicated signifance at the p −0.062 level comparing DFI from control to BCG treated patients to BCG + cells treated patients; a difference also was found when the control group was compared with the combined immunotherapy treated groups ( p = 0.061). When only patients with stage I disease were analyzed by trend analysis this difference was magnified in favor of the immunotherapy treated patients ( p = 0.027); analysis of the T 1 NoMo only groups revealed a similar result ( p = 0.042). No significant differences were seen when data were analyzed for survival. Moderately severe local inflammatory reactions occurred in 43% of patients who received BCG immunotherapy, requiring reduction in frequency of its administration. There were no adverse effects from the administration of allogeneic tumor cells. Pasteur BCC may prolong DFI in resectable lung cancer and warrants further investigation in clinical trials.

Acute myelomonocytic leukemia associated with paraproteinemia

A 49‐year‐old white woman with refractory anemia subsequently developed acute myelomonocytic leukemia with paraproteinemia 12 months later. The paraprotein was characterized as immunoglobulin G, type kappa, and the Bence Jones protein as free kappa chains. Further studies, including electron microscopy, cytochemistry, and immunofluorescence provided evidence for synthesis of the paraprotein, both in vivo and vitro, by the myelomonocytic leukemic cells.

Detection of Antibodies to Herpesvirus saimiri Late Antigens in Human Sera

One hundred fifty sera from handlers of squirrel monkeys and 100 sera from individuals who had never handled monkeys were tested by immunofluorescence for antibodies reactive to structural proteins of Herpesvirus saimiri (HVS). Eleven (7.3%) of the occupationally exposed group and 4 (4%) of the noncontact group were seropositive for HVS by immunofluorescence assay, and 10 of these 15 (6.7 and 2%, respectively) were also seropositive for either the major glycoprotein (140 kD) or the major capsid protein (160 kD) of HVS by radioimmunoprecipitation assay. Two sera from handlers of squirrel monkeys, however, recognized many different HVS structural antigens by immunoprecipitation, and it seems unlikely that they could also be cross-reactive antibodies. Since these two sera did not contain antibodies to HVS early antigens or to the nonstructural antigens present in infected owl monkey kidney cells, and follow-up sera collected from the same individuals several months later were negative for antibodies to HVS, these individuals do not appear to have been infected by the virus. The risk that HVS poses to humans appears to be very low.

Immunologic monitoring and immunotherapy in Ewing's sarcoma

SummarySerial immunological monitoring was performed on 31 patients with Ewings sarcoma who were on a randomized immunotherapy trial with BCG administered by dermal scarification with a Heaf gun. Patients were skin-tested for delayed hypersensitivity reactions (DCHR) to recall antigens and extracts of tumor cells, and with keyhole limpet hemocyanin (KLH). In vitro testing consisted of lymphocyte counts, percentages of cells forming rosettes with sheep erythrocytes at 29° C and at 4° C, and leukocyte migration inhibition to tuberculin (PPD) and to 3 M KCl extracts of tumor cells. At the time of diagnosis, nearly all patients had positive DCHR to mumps and streptococcal antigens and were negative to PPD. Neither the skin tests nor the lymphocyte counts at this time gave useful prognostic information. In tests during and after therapy, the patients who responded and remained free of detectable disease had a higher incidence of DCHR to KLH and of rosette values in the normal range than did the patients who developed recurrent disease. The BCG immunotherapy had no apparent effect on immunologic parameters except for conversion of reactions to PPD.

Variations in the immune response to herpesvirus saimiri in squirrel and rhesus monkeys

Humoral and cell-mediated immunity (CMI) to herpesvirus saimiri (HVS), an oncogenic lymphotropic herpesvirus, was studied in squirrel and rhesus monkeys. Natural antibody to HVS was found in five of six squirrel monkeys but there was no evidence of specific CMI directed against HVS. Rhesus monkeys did not show natural antibody or CMI against HVS antigens. Immunization with HVS, however, produced both antibody and specific CMI in the rhesus monkeys, but no CMI developed in the squirrel monkeys. These findings are important in the development of animal models for the treatment of tumors associated with lymphotropic herpesviruses.

Rapid Effects of BCG on T Cell Function in Cancer Patients

SummaryForty-four cancer patients were evaluated with multiple sequential tests to determine the effects of a single intracutaneous injection of BCG on the levels of total and high-affinity sheep erythrocyte-forming cells and on lymphoproliferative responses to a T cell mitogen (PHA) and antigens (MLC and PPD). The analysis of the data was complicated by considerable pretreatment variation in the results with some patients and by the lack of a consistent change in responses among all patients. However, exploratory statistical procedures made it possible to demonstrate that inoculation with BCG was associated with a ‘significant’ transient increase, on day 3 post-injection, in levels of total and active rosette-forming cells and in lymphoproliferative responses to PPD. The effects on responses to PHA varied with the level of pretreatment reactivity with patients with initial low responses showing a significant increase on days 3 and 7 and patients with initial normal responses showing a significant decrease on days 3 and 7. Patients and normal individuals receiving no injection, and patients receiving isotonic saline injections, showed none of these changes. Thus it appears that BCG may cause measurable changes in T cell levels and lymphoproliferation to T cell mitogen (PHA) and antigen (PPD).