Tumoricidal properties of rat peritoneal macrophages activated with various activators depend on nitrogen oxid synthesis.

Cytolytic activity of mineral oil elicited rat peritoneal macrophages activated by lipopolysaccharide (LPS) and/or rIFN-gamma, rIL-2, Zymosan and PMA (4-beta-phorbol-12-beta-myristate 13-alpha-acetate) was detected in the presence of various concentrations of L-arginine. This paralleled the NO2- production in the presence, but not in the absence, of L-arginine. Significant amount of NO2- was detected in the peritoneal macrophages cultured with 0.4 mmol of L-arginine 8 days and the last 24 h with LPS at a concentration of 1 microgram/ml. No significant differences were found between activated peritoneal macrophages obtained from normal (healthy) and/or from tumor bearing rats to induce tumoricidal activity and NO2- production under the same experimental conditions. The results showed that the major cytolytic mechanism against BP6-Tu2 and U 937 tumor cell lines is L-arginine-dependent nitrogen oxide synthesis of activated rat peritoneal macrophages.

Tumor necrosis factor as a potent effector molecule for tumor cell killing by activated rat peritoneal macrophages in vitro.

Tumor necrosis factor (TNF) sensitive (BP6-Tu2) and less or insensitive (B77, MC-1) target cell lines were used to examine the role of TNF in lytic phases of activated peritoneal macrophages and/or direct (murine)-TNF alpha cytolytic activity against these targets. Using antibody to murine TNF, it was shown that lysis of BP6-Tu2 cells was TNF-mediated. This macrophage lytic activity was markedly diminished or even abolished by anti-TNF alpha antiserum. Results indicated that TNF alpha is the critical effector of macrophage mediated killing of these targets. The tumoricidal activity and its level was dependent upon the length of cultivation of macrophages, induction period and concentration of activators. The findings not only support the thesis that macrophages possess various means of coping with tumor cells but also suggest that the mechanism becoming operative is determined predominantly by the pathway of macrophages activation and the properties of the tumor-cell type.

In vitro activation of tumoricidal properties of rat peritoneal macrophages using lipopolysaccharide.

The peritoneal macrophages from normal Lewis rats were characterized by their capacity to phagocytose, by the presence of nonspecific esterase and Fc receptors. In vitro, these macrophages were maintained in culture 7 and/or 21 days, respectively, and for the last 24 h (activation period) were cultured with 0.1-4.0 micrograms/ml of lipopolysaccharide (LPS) and were found tumoricidal against different rat fibrosarcomas, at a ratio of 50:1 (BP6-Tu2, MC-1 and B77). Macrophage-mediated tumor cytolysis was determined using 51Cr-release assay. The sensitivity of used tumors to macrophage-mediated cyto-toxicity was different. In vivo the transfer of the activated macrophages together with the mentioned tumor cells to rats inhibited the growth of tumors. The adoptive transfer of macrophages activated with "activators" might lead to a new kind of immunotherapy of neoplastic diseases.

[Antitumor immunity in patients with melanoma of the uveal tract]. / Protinádorová imunita u pacientov s melanómom uveálneho traktu.

Over the years 1977-1987 142 patients with malignant melanoma of the uvea were admitted to the Department of Ophthalmology, Teaching Hospital, Comenius University in Bratislava. Of the 119 patients treated by enucleation of the bulb 74.8% were alive after a mean follow up period of 5.9 years, whereas 25.2% of these patients died of consequences of metastases of the melanoma of the eye. The highest death rate was recorded in the first and second year following enucleation (57%) and then in the fourth year (17%). After the eighth year following enucleation of the bulb not a single case of death from consequences of metastases of melanoma was recorded. Eight patients with localization of the tumor in the anterior part of the uveal tract (iris, ciliary body) were treated by microsurgical removal of the tumor from the bulb. Two patients developed relapse of the tumor and the eyeballs had to be enucleated. After a mean follow up period of 6.6 years all these patients are alive and so far no signs of metastases have been observed. Moreover, the visual acuity has remained good in the patients whose eyes were not enucleated. Electrocoagulation was applied in six patients. Within a year the eyes had to be enucleated in three of them due to tumor progression. In two patients the growth of the tumor seems to have been stopped, yet regression of the tumor has not been observed. Complete healing was recorded only in one patient. After a mean follow up period of 4.5 years all the patients are alive without signs of metastases. A series of eight patients refused any kind of treatment of melanoma. Over a follow up period of 8.5 years six of them (75%) died of consequences of metastases within an average of 3.2 years from the establishment of diagnosis. Although the majority of these cases had small and middle sized melanomas, at present only two patients (25%) are still alive. In 127 patients with histologically proved malignant melanoma of the uvea the LAI test for the determination of uveal tumor antigen was also performed. The test was positive in 78.7% of the patients and negative in 23.3%. In a control group of patients with nontumorous eye diseases the LAI test was positive only in 2.9% and negative in 91.1%. Four months after enucleation of the bulb the LAI test was repeated and positivity was found to persist in 57.8% of the patients.(ABSTRACT TRUNCATED AT 400 WORDS)

[Detection of surface antigens in uveal melanoma using monoclonal antibodies]. / Detekcia povrchových antigénov u uveálnych melanómov pomocou monoklonálnych protilátok.

Monoclonal antibodies (MA) were obtained by fusion of a myeloma line from mice with spleen cells of mice previously immunized by live human cells of uveal melanoma of an epithelioid type (UMEL-H). From the cell fusion hybridomas were obtained which produced antibodies against immunized uveal melanoma cells. The binding capacity of MA was assessed by the sandwich radio-immuno (RIA) test using indirect immunofluorescence. Extensive specific tests revealed that MA are strongly bound to uveal melanoma, the cells of which were used to immunize the mice, but also fresh allogenic uveal melanoma cells UMEL-K which were also of the epithelioid type. A smaller binding activity was observed with the cell line of the uveal melanoma VUP-1 which was started 16 years ago and is formed by 92% by epithelioid cells. A smaller binding activity of MA was observed with the line of skin melanoma cells (HMB-2, B-HM8). The bond of MA with carcinomatous cells, fibroblasts, uveal and retinal cells from eyes of healthy humans was negative. The preliminary investigations indicate an antigenic homogeneity of tumour antigens of melanoma cells of the same histological type and the antigenic variability of uveal and skin melanomas.

Restoration of immune response by levamisole in B-77 virus induced tumor-bearing rats.

The immunorestorative effect of levamisole was observed in Lewis rats inoculated with a syngeneic B-77 virus induced tumor and immunized with sheep erythrocytes. In tumor-bearing rats the cell-mediated cytotoxicity, the delayed footpad reaction and the antibody production were suppressed. While the cytotoxicity suppressed in tumor-bearing rats was not restored by levamisole, delayed footpad reaction and antibody production were enhanced as compared with the normal reaction, or were restored to normal levels by levamisole, respectively. Tumor growth was significantly suppressed in rats treated with levamisole. The presence of tumor changed the uptake of 3H-uridine labeled lymphocytes in the spleen and peripheral blood. Restorative effect of levamisole on T lymphocytes was different in the studied organs.

Cell surface antigens of human uveal melanoma identified by monoclonal antibodies.

Hybridoma cells were derived from a mouse immunized with freshly obtained primary human uveal melanoma cells. Supernatants from the resultant hybridoma clones were screened for positive antibody binding to uveal melanoma cell membranes and negative binding to membrane preparations of fibroblasts, retinal and uveal cells of healthy donors using sandwich radioimmunoassay and indirect immunofluorescence. Extensive specificity tests showed that the antibodies produced by ten clones bound strongly to fresh, and/or short-time cultivated primary human uveal melanoma tumour cells (UMEL-H, UMEL-K). Weaker binding occurred in a human uveal melanoma cell line (VUP-1), and/or in human skin melanoma cell lines (HMB-2, B-HM8). Binding assay of carcinoma cells, fibroblasts, uveal and retinal cells was negative. Intensive screening of this type is now under way.

A preliminary report on monoclonal antibodies against human uveal melanoma.

Mouse Sp2/10 myeloma cells were fused with spleen cells from mice that had been immunized with freshly obtained primary human uveal melanoma cells. Hybrids that produced antibodies binding to the uveal melanoma cells, but not to fibroblasts, uveal or retinal cells of healthy donors, were cloned. Extensive specificity tests showed that the antibodies produced by the ten clones bound strongly to fresh or short-time cultures of primary human uveal melanoma tumor cells (UMEL-H, UMEL-K). Weaker binding occurred with a human uveal melanoma cell line (VUP-1), and with human skin melanoma cell lines (HMB-2, B-HM8), respectively. Binding assays with carcinoma cells, fibroblasts, uveal and retinal cells were negative. An intensive screening of this type is now under way.

Peripheral blood leukocyte adherence inhibition (LAI) reactivity before and during progressive growth of B77 tumor in rats.

Sequential changes in B77-rat fibrosarcoma (B77-RF) extract-induced leukocyte adherence inhibition (LAI) reactivity of peripheral blood leukocytes (PBL) were investigated in rats from the time of tumor inoculation through progressive growth and/or surgical excision of tumor. The time-course profiles of LAI reactivity of PBL in tumor bearing rats correlated with the stage of tumor growth. LAI reactivity of PBL in tumor bearing rats increased during 5-15 days after cell inoculation. Simultaneously, using in vitro 3H-uridine incorporation method for detection of T-cell population, the significantly increased number of immunocompetent lymphocytes was found. During progressive tumor growth (16 days and more after tumor cell inoculation) the LAI reactivity decreased. In this period moderate depletion of immunocompetent population of lymphocytes was observed. A significant decline in LAI reactivity was noted 10 days after a thorough excision of the tumors as compared to that prior to the surgical intervention. The kinetics of the observed time-course appearance of LAI reactivity and the demonstration of a rapid response following tumor challenge indicate further that LAI is a reliable in vitro measure of in vivo tumor immunity.

Further study of specific immunoreactivity in patients with malignant melanoma of uveal tissue by LAI assay.

Cell immunity was followed in patients with malignant melanoma of the uveal tissue with the aid of LAI (leukocyte adherence inhibition assay). The LAI test proved positive in 59 out of 65 patients with histological evidence of malignancy of the melanoma (90.7%). The test was repeated three to six months following excision of the tumor from the eye in 48 patients and in 19 of them (39.6%) it was found to be negative. All these patients have survived and after a mean lapse of 2.5 years following the surgical intervention no metastases have been observed. As against this, LAI positivity persisted in the remaining 29 patients (60.4%), with metastases present in the liver or the eyeball in 8 of them (27.6%) during the follow-up period (1-4 yrs). Out of 39 patients with the diagnosis of suspected malignant melanoma of the uveal tissue, the LAI test proved positive in 17 (43.6%), but as subsequently ascertained, 6 of these (15.4%) had benign choroid nevi. In 6 further patients with suspected malignant melanoma of the uvea in whom the LAI test had been negative, this disease was subsequently ruled out. From a control group of 101 patients with various non-tumorous diseases of the eyes, a false positivity of the LAI test was noted in only two (1.98%).

Study of cell-mediated immunity in B77 tumor bearing rats by 51chromium release assay.

Cell immunity was followed in Lewis rats with transplanted syngeneic B77 tumors, by means of the cytotoxic 51chromium release test. Lymphocytes from peripheral blood, or from regional lymph nodes of tumor bearing animals, cocultured for 24 h with target B77 cells had an enhanced cytotoxic effect on target cells in comparison with lymphocytes of control donors. This effect of the lymphocytes still increased during 48 h of coculture.

Application of the indirect tube LAI assay in the study of cell-mediated immunity in rats immunized with B77 tumor cells.

The indirect tube LAI (leukocyte adherence inhibition) test has revealed that peripheral blood lymphocytes of rats of the inbred Lewis strain immunized with liver B77 tumor cells incubated in the presence of a specific tumor extract, release a factor into the culture medium that inhibits adherence of normal syngeneic peritoneal cells. Supernatants in which lymphocytes of healthy - control animals had been incubated in the presence of the same extract (B77), or lymphocytes of immunized donors incubated in the presence of another type of tumor extract (MC-1) were devoid of such a property. This soluble factor was found in the supernatants only after 20 hours of incubation of sensibilized lymphocytes with a specific tumor extract. Th percentage of adhering cells decreased in dependence on the concentration of the soluble factor. The results are discussed in connection with questions relevant to the mechanism of LAI reactivity and the application of this phenomenon in studies on cell immunity.

Comparison of LAI reactivity in B 77 tumor bearing rats between, before and after surgical removal of tumors.

Using a new modification of the tube LAI test, LAI reactivity was followed in rats of the inbred Lewis (LW) strain with transplanted syngeneic B 77 tumors before and one month after a surgical removal of the tumors. A significant decline in LAI reactivity was noted after a thorough extirpation of the tumors as compared to that prior to the surgical intervention. The results have been compared and are here discussed with a view to a possible application of the LAI test in studies of antitumor immunity in patients with a malignant disease.

LAI reactivity in rats immunized with tumor cells.

Leukocytes of peripheral blood of F1 hybrid inbred strain of rats LW X AVN and rats of inbred Lewis strain, immunized for three consecutive weeks with increasing doses of live or dead MC-1 or B 77 tumor cells, incubated for 20 hours with specific tumor extract, showed a lower adhering ability (LAI 32.8 +/- 16.6, 44.4 +/- 14.0, 43.1 +/- 7.4%) than that of the same cell population cultured without a specific antigen. The nonspecific tumor extract did not produce any LAI reactivity (4.4 +/- 5.9, 5.8 +/- 8.2, 6.7 +/- 5.9%). The values of LAI leukocytes of the controls tested by both the antigens were concordant with those found in samples of the same cell population tested without any antigens. The discussion bears on a possibility of applying the 20-hour modification of the LAI test in studies of cell immunity in immunized patients.

Peripheral blood lymphocytes and EAC-rosette forming cells in B77 tumor bearing rats.

The percentage and absolute numbers of peripheral blood lymphocytes and EAC-rosette forming cells were evaluated in B77 tumor bearing rats. The peripheral blood lymphocytes were in high level even in rats bearing very small tumors; this result suggest the mobilization of lymphoyctes into peripheral blood in tumor bearing rats. The increase of EAC-rosette forming cells was accompanied with the growth of tumors.

Blocking factor in sera of MC1 and B77 tumor bearing rats detected by LAI assay.

A modified form of the leukocyte adherence inhibition assay (LAI assay) has been used to detect the blocking factor in serum of the Lewis (LW) inbred rats and the F1 hybrids of the inbred LW X AVN strains with transplantated syngeneic B77 and MC1 tumors. This study has indicated that the cell mediated antitumor immune response of rats immunized with living tumor cells and the blocking factor in serum of rats with progressively growing tumors may be demonstrated by the LAI assay with incubation time prolonged to 20 hr (LAI assay-IT20). In addition, the specificity of the blocking effect and the disappearance of blocking factor after surgical removal of tumors have also been demonstrated in this paper.

Detection of specific immunoreactivity by LAI assay in patients with malignant melanoma of uveal tissue.

To monitor the specific antitumor immunity to patients with malignant melanoma of the uveal tissue, use was made of the 20-hr modification of the leukocyte adherence inhibition (LAI) test. Simultaneously, changes in the proportionally of T (total and active) and B lymphocytes in peripheral blood were followed in the same patients by the E and EAC rosette formation test. Peripheral blood leukocytes from patients with malignant melanoma of the uvea, cultured in vitro for 20 hr with syngeneic and allogeneic tumor extract showed a significantly lower adhering capacity than the same cell population cultured without the specific tumor extract (21/22). A retest 3 months after tumor removal showed the percentage of positive cases to have declined (11/19). Leukocyte adherence of control donors, cultured with and without the tumor extract remained unchanged. A follow-up of immunocompetence in patients with malignant melanoma of the uvea, based on proportionally changes of total T, "active" T and B lymphocytes yielded information of an orienting value only. The results of investigation of the various cases proved to be considerably divergent and failed to present a reliable picture of the patient's immunological status.

Evidence of cell-mediated antitumor immunity in rats bearing syngeneic tumors by the leukocyte adherence inhibition test.

A modified form of the leukocyte adherence inhibition (LAI) test has been used to follow cell-mediated immunity (CMI) in rats of the Lewis inbred strain (LW), F1 hybrids inbred strains LW x AVN with transplanted syngeneic tumors B77, MC-1, and the random-bred Sprague Dawley strain with spontaneous REF tumor. Peripheral blood leukocytes of animals incubated witha specific tumor extract showed an adherence capacity by 30--40% lower than the same cell population cultured without this specific extract, or with a foreign nonspecific extract. The results are commented in connection with a possible application of this modified form of LAI test in studies of the immunological state of patients with a malignant disorder.