Macrophage activation for the production of immunostimulatory cytokines by delivering interleukin 1 via biodegradable microspheres.

Interleukin 1alpha (IL-1alpha), a pleiotropic cytokine with multiple anti-tumour activities, has been investigated in our laboratory for its potential to serve as an immunotherapeutic agent. In the present study, an attempt was made to direct IL-1alpha to macrophages, in order to induce their immunoregulatory activities. For that purpose, IL-1alpha was encapsulated within biodegradable poly(lactic/glycolic acid) microspheres, 1-5 microm diameter in size. The microspheres were efficiently taken-up by macrophages in culture and after intraperitoneal injection into mice. In culture, phagocytosis of the microspheres reached saturation within 3 h and there was no apparent effect of polymer type on the extent of uptake. In vivo uptake of human IL-1alpha-microspheres by the macrophages lead to cell activation, as evidenced by the enhanced production of murine IL-1alpha, IL-6 and IL-12. Control microspheres, containing bovine serum albumin, induced only background to low levels of cytokine production. These cytokines, when expressed by or secreted from macrophages, may stimulate in situ diverse immune and inflammatory responses, including T cell-mediated immune responses, such as the development of Th(1)cells and cytotoxic lymphocytes. Thus, directing IL-1alpha into macrophages, via the appropriate microspheres, may serve as a unique mean to activate these cells to participate in anti-tumour immune responses in situ.

Antitumor and immunotherapeutic effects of activated invasive T lymphoma cells that display short-term interleukin 1alpha expression.

Expression of cytokines in malignant cells represents a novel approach for therapeutic treatment of tumors. Previously, we demonstrated the immunostimulatory effectiveness of interleukin 1alpha (IL-1alpha) gene transfer in experimental fibrosarcoma tumors. Here, we report the antitumor and immunotherapeutic effects of short-term expression of IL-1alpha by malignant T lymphoma cells. Activation in culture of T lymphoma cells with lipopolysaccharide-stimulated macrophages induces the expression of IL-1alpha. The short-term expression of IL-1alpha persists in the malignant T cells for a few days (approximately 3-6 days) after termination of the in vitro activation procedure and, thus, has the potential to stimulate antitumor immune responses in vivo. As an experimental tumor model, we used the RO1 invasive T lymphoma cell line. Upon i.v. inoculation, these cells invade the vertebral column and compress the spinal cord, resulting in hind leg paralysis and death of the mice. Activated RO1 cells, induced to express IL-1alpha in a short-term manner, manifested reduced tumorigenicity: approximately 75% of the mice injected with activated RO1 cells remained tumor free. IL-1 was shown to be essential for the eradication of activated T lymphoma cells because injection of activated RO1 cells together with IL-1-specific inhibitors, i.e., the IL-1 receptor antagonist or the M 20 IL-1 inhibitor, reversed reduced tumorigenicity patterns and led to progressive tumor growth and death of the mice. Furthermore, activated RO1 cells could serve as a treatment by intervening in the growth of violent RO1 cells after tumor take. Thus, when activated RO1 cells were injected 6 or 9 days after the inoculation of violent cells, mortality was significantly reduced. IL-1alpha, in its unique membrane-associated form, in addition to its cytosolic and secreted forms, may represent a focused adjuvant for potentiating antitumor immune responses at low levels of expression, below those that are toxic to the host. Further assessment of the immunotherapeutic potential of short-term expression of IL-1alpha in activated tumor cells may allow its improved application in the treatment of malignancies.

Involvement of immune responses in the eradication of IL-1 alpha gene-transduced tumour cells: mechanisms of tumour rejection and immunotherapeutical implications.

We detected a strong correlation between the constitutive expression of IL-1 alpha and reduced tumorigenicity, using fibrosarcomas which produce the cytokine spontaneously (as an aberration of the transformation process) or upon gene transfer. In fibroblasts intracellular or membrane-associated IL-1 alpha is expressed, whereas the secreted form of the cytokine (IL-1 beta) is absent. Studies on the mechanisms of tumour regression of the IL-1 alpha producing fibroblastoid cell lines indicated that IL-1 alpha potentiates the development of tumour cell-specific CTLs, which are of importance for tumour eradication. It also appears that IL-1 alpha-induced enhanced helper T-cell activity provides auxiliary signals for the growth/development of CTLs. In addition, we observed a massive lymphocytic infiltrate in IL-1 alpha producing regressing tumours which ultimately replaces the tumour's mass. Non-adaptive effector cells, activated locally by IL-1 alpha expressing fibrosarcoma cells, were also shown to contribute, to some extent, to the eradication of IL-1 alpha expressing fibrosarcomas. Local IL-1 alpha expression potentiated antigen presentation, by the malignant fibroblasts as well as by tissue-resident antigen-presenting cells, and by this anti-tumour immune responses were further potentiated. Mice, in which IL-1 alpha producing tumours regressed, developed systemic immunity and rejected a challenge with a non IL-1 producing violent tumour cell line. It appears that endogenous IL-1 alpha, being a strong inducer of cytokine production, operates a whole cytokine cascade (such as IL-6, CSFs and prostaglandins). However, studies using clonal populations have indicated that IL-1 alpha is essential for fibrosarcoma eradication, whereas the other cytokines possibly amplify and sustain its action. We assume that most naturally occurring tumours are not constitutive IL-1 alpha producers, as it would be disadvantageous for the tumour to express a cytokine which increases its immunogenicity. However, IL-1 non-producing fibrosarcomas can be induced easily to express IL-1 transiently, by treatment with cytokines/LPS, and upon the induction of the cytokine they shift from progressor to regressor tumours. We also obtained positive immunotherapeutical effects when treating mice bearing IL-1 non-producing fibrosarcomas with cells from the same line induced in vitro to express IL-1 alpha. The results may shed light on a novel parameter affecting tumour-host interactions, namely cytokine expression by the tumorous cells, and may provide the basis for new immunotherapy protocols for fibrosarcoma management.

[Low-molecular peptides in food as factors altering the resistance of rats to emotional stress]. / Nizkomolekuliarnye peptidy pishchi kak faktory izmeneniia ustoichivosti krys k émotsional'nomu stressu.

The authors studied the resistance of animals to emotional stress when they were given in the diet, as a source of protein, casein and a protein fraction composed of a mixture of amino acids and low-molecular peptides (18 and 2.7%) obtained by reprocessing Saccharomyces cerevisiae yeasts, biomass. The ECG, rheovasogram, systolic arterial pressure, and respiratory rate were studied in rats given a diet containing 18 and 2.7% low-molecular peptides (controls) and in the same animals after 24-hour immobilization. Addition of low-molecular peptides in the indicated concentration to the diet reduced the resistance of animals to emotional stress.

[Computer classification of the fusion profiles of human lymphocyte interphase chromatin]. / Klassifikatsiia profilei plavleniia interfaznogo khromatina limfotsitov cheloveka, poluchennaia s pomoshch'iu EVM.

AO fluorescent microscopy coupled with thermal denaturation of DNP cells as modified by the authors was used to study the structure of lymphocyte interphase chromatin from 164 normal persons. Analysis of the data processed by means of a Sperry Univac Computer 90/30 has demonstrated that in 40% of the cases, the melting profiles of DNP cells from normal persons represent, irrespective of the sex, a complicated but consistently repeated curve with 6 peaks at certain temperatures, i.e. that different test subjects have a pronounced "similarity" in the characteristics discussed. In the remaining cases, there have been recorded diverse but consistent deviations that correlated with the sex of the test subjects. No peak at a temperature of 85 degrees and its appearance at 82 degrees C were the most frequent deviations seen in the male group. There have been obtained altogether 5 subgroups for females and 7 subgroups for males. Thus an attempt has been made to classify for the first time the melting profiles of DNP cells from normal persons with the use of computer.