Higher ADCC of murine peritoneal cells after immunization with allogenic tumor cells as compared with stimulation by adriamycin, BCG, and thioglycolate

Normal peritoneal cells or spleen cells from C57BL mice could not lyse SRBC in an ADCC assay. After intraperitoneal injection of Adriamycin, BCG or thioglycolate the ADCC of peritoneal cells toward antibody-coated SRBC was elevated to 30% in contrast to the ADCC of spleen cells. However, peritoneal cells but not spleen cells of mice immunized with allogenic tumor cells (DBA SL2) showed ADCC levels at least two times higher than the levels observed after stimulation by other agents. Maximal ADCC levels (55.8%) were observed 10 to 15 days after immunization. Direct cytotoxicity towards SRBC increased to a maximum of 17.7% at 9 days after immunization. The effector cells in this system are thought to be macrophages, for ADCC activity was only present in the plastic-adherent cell fraction. Cell to cell contact was necessary for ADCC to occur; nonsensitized erythrocytes were not lysed when added to a mixture of effector cells and sensitized erythrocytes. Concentrations of antibody of 1 pg/ml were sufficient to induce ADCC, and effector cell to target cell ratios could be as low as 0.05. The finding that macrophages of mice immunized with allogenic tumor cells exhibit higher ADCC levels than macrophages elicited in other ways can contribute to the investigation of combined cancer therapy with antibodies and biological response modifiers.     

Interactions ofCandida albicans with bacteria and salivary molecules in oral biofilms

The yeastCandida albicans coaggregates with a variety of streptococcal species, an interaction that may promote oral colonization by yeast cells.C. albicans andCandida tropicalis are the yeasts most frequently isolated from the human oral cavity and our data demonstrate that both these species bind toStreptococcus gordonii NCTC 7869 while two otherCandida species (Candida krusei andCandida kefyr) do not. Adherence ofC. albicans was greatest when the yeast had been grown at 30° C to mid-exponential growth phase. For 21 strains ofC. albicans there was a positive correlation between the ability to adhere toS. gordonii and adherence to experimental salivary pellicle. Whole saliva either stimulated or slightly inhibited adherence ofC. albicans toS. gordonii depending on the streptococcal growth conditions. The results suggest that the major salivary adhesins and coaggregation adhesins ofC. albicans are co-expressed.     

The VX2 carcinoma in the rabbit auricle as an experimental model for intra-arterial embolization of head and neck squamous cell carcinoma with dextran microspheres

A head and neck cancer model is developed using the VX2 carcinoma cell line injected s.c. in both ears of New Zealand White (NZW) rabbits. The study is focused on the effects of intraarterial embolization of the carcinomas with a new type of dextran hydrogel microspheres. During the phase of exponential growth the tumour-surface doubling-time was 7.1+/-2.0 days. Standard deviation in growth of the tumours was significantly larger between separate animals than between tumours growing in the left and right auricle of each individual animal (2.0 versus 0.65 days). A fresh cell suspension containing at least 10 x 10(6) vital tumour cells was necessary to yield a tumour-take of 85%. The caudal auricular artery perfuses the caudal half of the external ear and is very suitable for macroscopic cannulation. Histological evaluation shows, that the use of dextran hydrogel microspheres of at least 25 microm in combination with ligation of non-tumour perfusing branches of the central auricular artery yields diffuse embolization of the VX2 carcinoma. This tumour model can be of use in further studies to optimize particle size and dosage for embolization as well as to evaluate the effect of different anti-neoplastic drugs, slowly released by controlled degradation of dextran microspheres.     

Physiological routes from intra-uterine seminal contents to advancement of ovulation

Whole boar semen or seminal plasma has been demonstrated to advance the time of ovulation in gilts. As a means of clarifying this influence, the contribution of uterine lymphatics and their white cell populations has been examined. After duct visualisation with Evan's blue, lymph was sampled from a mesometrial vessel in eight pre-ovulatory gilts whose uterine lumen was infused simultaneously with whole semen in one ligated horn and saline in the contralateral ligated horn. Lymph was collected from cannulated vessels for periods of up to four hours under general anaesthesia. Thereafter, mesometrial lymph nodes, utero-tubal junction and uterine wall tissues were sampled. The proportion of nucleated cells in the sampled lymph increased towards the end of the collection period, but erythrocytes were found in all instances preventing a meaningful differentiation and identification of leukocytes. Prominent uterine lymph nodes were present in the mesometrium on both sides of the reproductive tract in 7 of 10 gilts. Differences in cellular contents were demonstrated between the side of the tract infused with semen and that infused with saline control. Two of 4 gilts had lower values for CD4 (Cluster Differentiation) and 3 of 6 gilts higher values for MHC II (Major Histocompatibility Complex) markers on the side challenged with semen. In contrast, values remained constant for CD8 but ranged widely for CD18. Immunohistochemical analysis of uterine tissue samples for MHC II+ cells revealed significant differences (P < 0.05) between the control and semen-treated ligated portions of the horns, as well as between the tissue sample of uterine wall and that from the utero-tubal junction, but there were no significant differences for CD4+ cells. It therefore remains plausible that semen-induced cytokines in the uterine lymph undergo counter-current transfer to the ipsilateral ovary and accelerate the final maturation of pre-ovulatory Graafian follicles.     

Production of specific macrophage-arming factor precedes cytotoxic T lymphocyte activity in vivo during tumor rejection

Summary Recently we published a hypothesis on the immunological events occurring during tumor rejection. One of the implications of this hypothesis is that specific macrophage-arming factor (SMAF) is produced early during the initiation of the immune response, whereas the classical cell-mediated immune response components, such as cytotoxic T lymphocytes (CTL), are produced later, that is, during the amplifier-effector phase. In this paper we establish the kinetics of the induction of (a) lymphocytes producing SMAF and (b) CTL. Groups of DBA/2 mice were injected i.p. once, twice or three times with irradiated and/or non-irradiated syngeneic SL2 tumor cells, the injections being given at intervals of 10 days. After each of these injections the production of SMAF and the expression of CTL activity were established. The results showed that in the peritoneal cavity SMAF-producing lymphocytes appeared earlier than cytotoxic lymphocytes (CTL). In addition, it was shown (a) that SMAF does not interfere with the in vitro cytotoxicity expressed by CTL and (b) that in addition to CTL memory cells, SMAF-producing memory cells were also induced after injection of syngeneic tumor cells. These data support the hypothesis that SMAF is involved in the early phase of the cellular immune response against tumors, whereas CTL are induced later.     

Eradication of tumor cells after injection into immunized hosts compared with the eradication of tumor cells after transfer of immune peritoneal exudates into tumor-bearing recipients

Summary DBA/2 mice were immunized against the syngeneic SL2 lymphoma by two or five injections with irradiated lymphoma cells given IP or SC, respectively. The antitumor efficacy induced in immunized mice was tested by (a) IP injection of the immunized mice with nonirradiated tumor cells and (b) transfer of the total immune peritoneal exudate, the cellular fraction only, or the cell-free fraction only, IP into tumor-bearing recipients, or (c) tumor neutralization tests (Winn assay). It was shown that immunized mice were able to reject 5×10⁷ SL2 tumor cells (8 of 14 mice survived >100 days), while in most transfer experiments 2×10⁵ SL2 cells could be eradicated. In the tumor neutralization experiments a number of 10⁶ SL2 cells were eradicated. When the immune exudates were given before the inoculations of SL2 tumor cells the number of survivors increased significantly. Further, it was shown that the cellular fraction is the major contributor to the antitumor effect in the transfer experiments, since there was no significant difference in tumor eradication after injection of a complete immune exudate and after injection of the isolated cellular fraction. Injection of the noncellular fraction had no measurable antitumor effect. An increase in the number of injections with total peritoneal exudates from immunized mice did not result in an increase in tumor eradication in the tumor-bearing recipients.Extra stimulation (IP) of immunized mice with 10⁴ nonirradiated cells 6 days after the last immunization resulted in an increase of the antitumor efficacy of these peritoneal exudates of these mice when collected 4–24 h after this stimulation. Extra stimulation with 10⁶ irradiated cells had no measurable effect.     

Critical factors for liposome-incorporated tumour-associated antigens to induce protective tumour immunity to SL2 lymphoma cells in mice

Physical and immunogenic properties of reconstituted membranes designed for the presentation of tumour-associated antigens (TAA) to the immune system are described. Proteins and lipids of crude membranes of SL2 murine lymphosarcoma cells were partially solubilized with octylglucoside. Reconstituted membranes, consisting mainly of unilamellar vesicles with a diameter of 0.03–0.15 μm, were formed by detergent removal and were purified by floatation in a discontinuous sucrose gradient to remove non-lipid-bound protein. Subcutaneous immunization of syngeneic mice with reconstituted membranes or with purified reconstituted membranes induced protection against an intraperitoneal challenge with 10³ viable SL2 cells. Reconstituted membranes were more immunogenic than crude membranes in immunoprotection experiments when compared on the basis of protein dose. Detergent removal was required to obtain an immunogenic presentation form of SL2 membrane antigens and to avoid toxicity associated with the detergent. Reconstitution of SL2 membranes in the presence of exogenous phospholipid slightly increased the fraction of protein that associated with the reconstituted membranes. However, the immunogenicity of the solubilized membrane TAA was not significantly affected by the presence of exogenous phospholipid. The reconstitution procedure described may be useful in identifying membrane factors required for the induction of immune responses against TAA. The versatility of the system may be employed to develop safe alternatives for whole-cell vaccines.     

Cell surface glycoconjugates as possible target structures for human natural killer cells: evidence against the involvement of glycolipids and N-linked carbohydrate chains

Natural killer (NK) cells can spontaneously kill various malignantcells, but the susceptibility towards NK cells differs greatlyamong different types of tumour cells. The molecules, whichare recognized by NK cells, have not yet been identified, butthere is ample evidence that target cell surface glycoconjugatesare involved in the interaction with NK cells. In this report,we show that the recognition of K562 target cells by human NKcells depends on the presence of protein-bound determinants,implying that glycolipids are not the primary target structureson K562 cells. The NK susceptibility of K562 cells was not alteredby enzymic removal of various cell surface carbohydrates oroligosaccharides, mostly related to N-linked carbohydrate chains.Treatment of K562 cells with 1-deoxynojirimycin and 1-deoxymannojirimycin,inhibitors of N-glycan processing, resulted in drastic alterationsin the carbohydrate phenotype of the cell surface, as couldbe shown by flow cytometric analysis of the lectin-binding propertiesof the cells. Despite these clear changes in N-glycosylation,the NK susceptibility of K562 cells remained unaffected. Summarizing,the results described in this report show that potential targetstructures for NK cells are protein bound, but the involvementof a specific (N-linked) carbohydrate determinant in the interactionbetween NK cells and target cells could not be established. cell adhesion molecules cell—cell interaction cell surface glycoconjugates natural killer cells target structures