Studies on the heterologous immunogenicity of a methanol-insoluble fraction of attenuated tubercle bacilli (BCG). II. Protection against tumor isografts.

A methanol-insoluble residue (MER) of phenol-killed attenuated tubercle bacilli (BCG), which has been reported previously to be capable of evoking heightened resistance to infection with antigenically unrelated microorganisms, was found to affect as well the resistance of highly inbred mice against tumor isografts. In most instances, the MER evoked heightened resistance against the tumor implants, but heightened susceptibility was the effect induced against two of the tumors tested, and no effect was elicited against one neoplasm. It is suggested that the heightened susceptibility occasionally produced by pretreatment with MER may also be of immunological nature, i.e. immunological enhancement. Treatment with MER was more effective when administered some time before tumor challenge than when given simultaneously with, or after, tumor implantation. The protective effects manifested against some tumors were of a high order, a significant number of animals rejecting the neoplastic implants, and were displayed even when several months elapsed between treatment and challenge. Living BCG and intact phenol-killed bacilli also evoked heightened resistance against some of the tumors tested, and in one experiment living BCG proved effective whereas MER did not. On the whole, however, MER was the most active (and least toxic, as shown previously) of the several tubercle bacillus preparations tested. MER elicited heightened reactivity against first transplant generation tumors as well as against tumors maintained for considerable periods of time by repeated animal passage, and against spontaneously arising as well as against induced neoplasms. The experimental parameters necessary to demonstrate maximal effects varied somewhat from tumor to tumor. In general, however, single intraperitoneal injections of small quantities of MER, of the order of 0.25 to 1.0 mg, afforded the best protection.

Immunology of the mouse mammary tumor virus: comparison of mammary tumor virus with the agent found in C3Hf/Crgl mice.

C3Hf/Crgl mice, containing no biologically active mammary tumor virus, nonetheless possess both type-B virus particles and a component which cross-reacts serologically with the mammary tumor virus. Neutralization studies and the immunodiffusion assay for measuring the antigenicity of the mammary tumor virus indicate that transfer of the virus particles from C3Hf to BALB/cCrgl mice is accompanied by transfer of the cross-reacting antigenic component.

In vitro induction of cell-mediated immunity to murine leukemia cells. III. Effect of methanol extraction residue fraction of BCG on the generation of cytotoxic lymphocytes against leukemia.

Effects of the methanol extraction residue (MER) fraction of tubercle bacilli (BCG) on the generation of cytotoxic lymphoid cells were studied in vitro with the use of unidirectional mixed lymphocyte-tumor cell cultures. These cultures consisted of splenocytes of lymph node cells from normal donor C57BL/6, BALB/c, and strain A mice and mitomycin C-inactivated leukemia cells of both syngeneic and allogeneic origin. Addition of small amounts of MER (0.2-5 microgram/ml) to the cultures potentiated appreciably the elicitation of cytotoxic reactivity (as measured by the 51Cr-release assay) of the sensitized cells, whereas higher quantities (10-40 microgram/ml) had a strong suppressive effect. MER also induced some cytotoxic capacity in normal murine and human lymphoid cells not exposed to specific tumor cell stimulation. The stimulatory and suppressive effects were noted only when MER was present during the initial 24-48 hours of the 6-day culture. With the nylon wool fractionation technique, it was apparent that MER affected primarily the nonadherent cell population. MER could also prevent the generation of nonspecific suppressor cells by splenocytes maintained for 3-6 days in tissue culture.

Effects of methanol extraction residue and therapeutic irradiation against established isografts and simulated local recurrence of mammary carcinomas.

Female BALB/c mice carrying established isografts or simulated local recurrence implants of 2 rapidly growing mammary adenocarcinomas were treated either by injection of the methanol extraction residue (MER) fraction of killed Bacillus Calmette-Guérin organisms (given s.c. or into the tumor) or by focal X-irradiation or by both. None of the modalities of therapy effected cures, but in many instances there was a significant retardation of tumor cevelopment and prolongation of the lives of the mice. Administration of MER alone offered protection in a number of cases but less often than the other forms of treatment. Combined therapy with MER and irradiation was, on the whole, the most successful therapeutic intervention. MER or irradiation administered alone enhanced the neoplastic process only on rare occasions; this appeared to be the case even more infrequently with combined treatment. MER was most likely to be effective alone or in combination when small quantities were used and when only 1 treatment or 1 cycle of combined therapy was given. The therapeutic action of MER was not dependent on direct introduction of the agent into a neoplastic focus; s.c. administration distal to the tumor site was almost always at least as satisfactory as injection directly into the tumor mass and indeed was often more efficacious.

In vitro induction of cytotoxic effector cells against human neoplasms. I. Sensitization conditions and effect of cryopreservation on the induction and expression of cytotoxic responses to allogeneic leukemia cells.

Peripheral blood lymphocytes (PBL) from normal human donors were sensitized in vitro against allogeneic human acute myelocytic leukemia (AML) cells by means of an unidirectional mixed lymphocyte-tumor cell culture (MLTC) technique. The cytotoxic responsiveness of the sensitized lymphocytes, as determined in vitro by the 51Cr-release assay, varied among individual lymphocyte donors and was greatly dependent on the sensitization culture conditions. Induction of cytotoxic effector cells was augmented appreciably by adding to the cultures minute amounts of the immunopotentiating agent MER-BCG. Responding lymphocytes and stimulating leukemia cells cryopreserved for several weeks in liquid nitrogen were as effective as fresh cells in generating effector lymphocytes; the cytotoxic capacity of already sensitized lymphocytes was fully retained by cryopreservation. The implications of these findings for possible clinical employment of in vitro sensitized lymphocytes in adoptive immunotherapy of cancer are discussed.

Chemoimmunotherapy of syngeneic mouse mammary carcinomas employing methanol extraction residue.

Isografts of two mammary carcinomas, one spontaneously arising in a BALB/cfC3H female infected with MTV and one free of MTV (tumor D7T4S), were removed surgically from Balb/c (MTV free) female hosts, and fragments of each tumor were immediately reimplanted in situ (simulated local recurrence challenge). The animals were then subjected to treatment with the MER fraction of tubercle bacilli, with one of three chemotherapeutic drugs (5-FU, cyclophosphamide, and methotrexate), or with both MER and one of the chemotherapeutic agents (chemoimmunotherapy). The incidence of progressively developing recurrence tumors and the longevity of the animals were determined. The therapeutic effects of treatment with MER alone were ascertained by comparing groups of mice that received the fraction by various schedules with saline-injected control groups; the efficacy of chemoimmunotherapy was assassed by comparing groups that received MER plus one of the drugs with groups subjected to drug intervention by itself. MER administered alone did not reduce the incidence of recurrent tumors but was consistently efficacious in prolonging the lives of animals challenged with the MTV (+) carcinoma, although considerably less so in animals tested with the weakly immunogenic tumor D7T4S. A negative effect by MER on tumor frequency did not occur and was seen only once with regard to host life duration. Combined intervention with MER and 5-FU proved to be significantly and consistently superior to similar treatment with only 5-FU in animals challenged with the MTV(+) carcinoma. No such additive action by MER plus 5-FU was seen in mice challenged with D7T4S, however, nor did the other two chemoimmunotherapeutic regimens differ significantly in therapeutic efficacy from the corresponding chemotherapy alone in most of the trials with both tumors.

Classification of cancer chemotherapeutic drugs according to their ability to potentiate contact sensitivity to dinitrofluorobenzene.

The anticancer chemotherapeutic drugs cyclophosphamide (CY), melphalan (L-phenylalanine mustard; L-PAM), 5-fluorouracil (5-FU), methotrexate (MTX), and daunorubicin (DAU) were tested for ability to potentiate contact sensitization (CS) to dinitrofluorobenzene (DNFB) in mice and to inhibit induction of unresponsiveness to such sensitization by preceding exposure to dinitrobenzenesulfonate (DNBS). Administration of CY and L-PAM but not of 5-FU, MTX, and DAU, after DNBS treatment (and before DNFB sensitization) reduced the degree of unresponsiveness effected by DNBS. It is suggested that the test system of CS might be utilized for screening the ability of cancer chemotherapeutic agents to act as nonspecific immuno-modulators.

Immunological reactivity to a mycobacterial fraction is associated with nonspecific suppression of immunological responsiveness in vivo.

Previously reported studies revealed that spleen cells from BALB/c mice immunized against a methanol extraction residue (MER) fraction of tubercle bacilli are defective in the in vitro generation of antibodies to SRBC and in allogeneic responsiveness against C57BL spleen cells. We now show that mice repeatedly immunized with MER also exhibit a depressed capacity to respond to antigenic stimulation in vivo. Thus mice repeatedly injected with MER were impaired in their ability to react to antigenic stimulation by SRBC and by C57BL spleen cells. Impairment in the response to SRBC immunization was expressed at the level of delayed-type hypersensitivity (DTH) as well as of antibody production. The response of MER hyperimmunized mice to contact sensitization with dinitrofluorobenzene (DNFB) was not impaired, but the lymph node cells of DNFB-sensitized animals had a depressed ability to respond to in vitro stimulation by the monovalent hapten dinitrobenzene sulfonate (DNBS). The present findings indicate that extensive exposure to an immunogenic immunomodulating mycobacterial fraction can lead to a depressed responsiveness to unrelated antigenic stimulation.

Studies in psychoneuroimmunology: psychological, immunological, and neuroendocrinological parameters in Israeli civilians during and after a period of Scud missile attacks.

Twenty-two male volunteers in Jerusalem were subjected to a battery of psychological tests at the height of the Iraqi Scud missile attacks on Israeli cities during the 1991 Persian Gulf War and again after the cessation of hostilities. Venous blood samples were taken at each time point. The separated mononuclear cells and plasma were cryopreserved, and a spectrum of immunological and neuroendocrine assays were performed on the preserved samples. Psychological testing indicated levels of anxiety were higher during the war than they were after the war ended, and both anxiety and anger during the hostilities were significantly elevated in comparison with prewar data. During the war, specific war-related pressures were greater than everyday pressures, and problem-focused coping was more evident than emotion-focused coping. Natural-killer cell activity and cell-mediated lympholysis were significantly elevated during the war, as were plasma levels of adrenocorticotrophic hormone, neurotensin, and substance P. The only biological test parameter found to be reduced during the war period was mononuclear cell thymidine incorporated in nonstimulated cultures.

Patterns of fibronectin deposition in normal and neoplastic fibroblasts and mammary tissue.

Deposition of fibronectin (FN) was studied by indirect immunofluorescent staining, employing monospecific rabbit anti-human FN antibody, in the following 3 systems: cultures of cells derived from Rous sarcomas of chickens and of normal chick fibroblasts; frozen sections of chicken Rous sarcomas; and frozen sections of mouse and human mammary tissue, normal and neoplastic. The normal fibroblasts produced a well-oriented, delicate, fibrillar FN network. The sarcoma cells in culture also formed a FN-positive matrix, but one very different in appearance, consisting of coarse, unoriented strands. FN was also present abundantly between the cells of the sarcomas in vivo. In normal mammary tissue, FN was localized predominantly in the basal lamina region, well delineated from the surrounding FN-reactive stroma. The epithelial cells were generally, but not always, FN-negative. In apparently normal tissue areas of the breasts of mammary carcinoma patients, epithelial cells with highly reactive cell peripheries were frequently seen; the stroma surrounding the positive cells was often poor in FN and the basal lamina region lacked the substance. Mammary carcinoma cells were almost always negative, but it was characteristic of these tumours that the surrounding stroma displayed FN richly.

Immunofluorescent characterization of fibronectin, laminin, and keratin in normal and neoplastic human mammary epithelial cells in culture and in breast tissue sections.

Employing indirect immunofluorescent staining, primary and secondary serum-free cultures and frozen sections of human mammary tissue, normal and neoplastic, were examined for the presence and distribution of fibronectin (FN) and keratin, and frozen sections also for laminin (LM). The epithelial cell purity of the cultures was confirmed by the observation that all cells stained with anti-keratin antibody. In confluent cultures, FN was absent at the apical cell surface, and was seen as a fibrillar matrix exclusively beneath the epithelial monolayer, at the cell-substratum interface. No differences were noted between normal and neoplastic cells in vitro. In sections of normal breast tissue, FN was localized in the basement membrane zone (BMZ) and in the connective tissue stroma. A distinguishing feature of the neoplastic tissue was the considerably more intensive anti-FN immunofluorescence of the stroma. In normal tissue sections, LM was present exclusively in the BMZ, where it formed a continuous, well-delineated, smooth line; this line was found to be distorted, interrupted, and sometimes entirely absent in the neoplastic tissue. The cytoplasm of all cultured cells, neoplastic as well as normal, exhibited a dense network of keratin filaments that was especially prominent around the nucleus. In the sections, keratin was ubiquitously present in the epithelial cells, predominantly along the interior border of the surface membrane; in the neoplastic tissue, this pattern was markedly disorganized, and some of the cells failed to express the substance.