Persistence of SCE-inducing damage in mouse embryos and fetuses following superovulation.

The frequency of sister-chromatid exchange (SCE) was analyzed in postimplantation embryos of superovulated Swiss and C57BL/6J Han mice. 12- and 16-day-old embryos, analyzed for SCE in vitro as whole embryo primary cell cultures, displayed an average increase in SCE of about 50%. Although an increased frequency of SCE cannot be used for the prediction of specific effects on the health of an individual, damage and possible repair of DNA which provide opportunities for modification of chromosomal structure and function may be present.

In vitro SCE discrepancy between embryonic and extraembryonic mouse tissues.

In vitro sister-chromatid exchange (SCE) background levels and cytokinetics were compared in embryonic (whole embryo cell suspensions) and extraembryonic (yolk sac and amnion, placenta) cells of inbred and outbred strains at various gestational stages (days 12-17). Results indicate a tissue origin (embryonal, extraembryonal) related variation in the formation of baseline SCE frequencies and cytokinetics. The significant higher SCE levels in extraembryonic tissues (maximum increase of 2 X the background values of the embryo cells) were independent of mouse strain and gestational stage. An average of 4-5 SCEs/cell in embryo cells is contrasted by 7-9 SCEs/cell in extraembryo cells. Mitotic index was generally lower and average generation time longer (by 2-3 h) in extraembryonic tissue cells. No significant differences in SCE frequencies and no changes in cytokinetics were detected at the BrdU concentrations used (1.2-4.8 micrograms/ml). The reason for the inter-tissue differences in baseline SCE is still not clear.

A method for analysing sister-chromatid exchange in mouse preimplantation embryos.

Analysis of sister-chromatid exchange (SCE) has been shown to be a sensitive and reproducible method for detecting the action of mutagens and carcinogens. We have succeeded in establishing a reliable technique which allows to perform SCE in preimplantation embryos in order to make the pre-uterine stages of development accessible to routine detection of DNA damage. Using the mouse strain and technique described, approximately 30-40% of mice will mate successfully after synchronization and spontaneous ovulation. From 3 pregnant females, about 30 four- to eight-cell embryos will be obtained, representing one experimental group providing approximately 50-80 two-S-phase labelled metaphases with a SCE frequency baseline below 6 exchanges.

Abnormal development and transport and increased sister-chromatid exchange in preimplantation embryos following superovulation in mice.

Both sister-chromatid exchange (SCE) response and embryonic development and transport in preimplantation embryos were evaluated on day 3 of gestation (vaginal plug = 1) of superovulated Swiss mice. Superovulation was found to have significant effects on number of preimplantation embryos (increase), embryo localization (accelerated transport), cleavage rate (advanced development) and abnormality rate (misshaped, fragmented, dead embryos). Superovulated 4- and 8-cell embryos collected from oviducts and uteri and incubated in vitro with 5-bromodeoxyuridine (BrdU) displayed up to 4 times higher SCE frequency than spontaneously ovulated embryos. This increase is independent of stage of development and location at the time of embryo collection. The results indicate that superovulated embryos may have induced DNA lesions.

Influence of thia-benzimidazole Wy 18251 of cancer patients' lymphocyte reactivity.

The influence of thiazolobenzimidazole Wy 18251 on reactivity of cancer patients' lymphocytes was investigated. Doses of 0.2 to 20 micrograms/ml added to lymphocytes for 72 hrs did not influence spontaneous 3H-thymidine uptake nor PHA-induced blastogenic response. 50 micrograms/ml of Wy 18251 significantly enhanced blastogenic response of lymphocytes in 5 out of 8 patients. Augmentation of response to mitogens was mainly observed when PHA was added in a concentration leading to suboptimal stimulation. These results suggest that Wy 18251 is active on suboptimal stimulated lymphocytes but ineffective on unstimulated or optimal stimulated human lymphocytes. Further studies are required to give insight in this mechanism of stimulation.

Suppression of natural killer cell activity by adherent effusion cells of cancer patients. Suppression of motility, binding capacity and lethal hit of NK cells.

Adherent cells from carcinomatous pleural effusions of lung cancer patients were tested for their ability to suppress natural killer (NK) cell activity, and the mechanism involved in the suppression of NK cell activity was determined. Adherent effusion cells (AEC) were isolated from malignant pleural effusions of patients by centrifugation discontinuous Ficoll-Hypaque gradients and adherence to serum-coated plastic dishes, and large granular lymphocytes (LGL) were purified from the peripheral blood of normal individuals by centrifugation on discontinuous Percoll gradients and further depletion of high-affinity sheep erythrocyte rosette formation. LGL-mediated lysis of K562 cells was suppressed when LGL were cultured with AEC for 20 h, then washed and tested in a 4-h 51Cr release assay. More profound suppression of NK cell activity was observed when cytotoxicity was assayed in flat-bottomed wells rather than in round-bottomed wells. Cytotoxicity assays conducted at the single cell level in agarose revealed that the frequency of LGL binding to K562 cells and of dead conjugated target cells was reduced after overnight contact with AEC. In agarose microdroplet assays, functional LGL from normal donors exhibited definitive motility, expressing polarized shape. In contrast, a small number of LGL with non-polarized configuration migrated from the agarose droplet after overnight culture with AEC. These results indicate that functionally suppressed NK cells lose their motility, binding capacity and killing activity, which could be responsible for the suppression of NK cell activity by AEC.

Chemotactic properties of partially purified human epidermal cell-derived thymocyte-activating factor (ETAF) for polymorphonuclear and mononuclear cells.

Human epidermal cell thymocyte-activating factor (ETAF) derived from either normal epidermal cells or a squamous cell carcinoma cell line has recently been shown to be a low m.w. protein that is indistinguishable from human macrophage-derived interleukin 1 (IL 1). As with human IL 1, human ETAF elutes from Sephadex S-200 gel in two major peaks at m.w. 70,000 to 40,000 and m.w. 25,000 to 12,000. Rechromatography of the higher m.w. fraction of ETAF yielded some of the lower m.w. activity, and chromatofocusing of high m.w. ETAF yielded the same three isoelectric points as the lower m.w. ETAF. Partially purified human ETAF as well as IL 1 were chemotactic for polymorphonuclear and mononuclear leukocytes. In addition, exposure of PMN to ETAF stimulated increased metabolic activity as demonstrated by greater reduction of intracellular nitroblue tetrazolium. Therefore, this study lends further support for an important role of ETAF in the pathogenesis of inflammatory skin diseases.

Plasmapheresis in patients with advanced malignant disease: a pilot study.

Plasmapheresis has been performed in 16 patients with advanced malignant disease (12 malignant melanoma, 2 breast cancer, 2 colon cancer). Up to 3,000 ml of plasma (range 1,650-3,000 ml) have been replaced by one single plasma exchange. Side effects observed in about 50% of the patients were mainly nausea and chills. Serum proteins including acute phase reactants were monitored before and 2, 4, and 7 days after plasma exchange. A transient change of these proteins was observed in the first days, but, later on, pretreatment values were always retained. Peripheral blood lymphocyte blastogenic response to mitogens was increased in 5 out of 9 patients 7 days after plasma exchange. In 7 out of 9 patients pretreatment serum was found inhibitory to autologous lymphocyte reactivity to mitogens. Furthermore, plasmapheresis was found to decrease significantly the blocking effect of patients' sera on normal donor lymphocyte reactivity. Plasmapheresis was found tolerable in all patients treated, but without any clinical efficacy. Further studies are warranted to establish the therapeutic value of plasma exchange in patients with advanced malignancies.

Variability in the preventive action of the B-blockers on genetic hypertension in the rat.

Changes in arterial blood pressure were compared in young male SHR rats untreated or treated orally with a beta-blocker for 5 days out of 7 from the 5th to 9th week of life. Propranolol, acebutolol and practolol, at a daily dose of 50 mg/kg, prevented the occurrence of hypertension, not only during the 4 weeks of treatment, but also during 6,9 and 11 weeks respectively after cessation of treatment. Alprenolol and metoprolol (50 mg/kg/day) and atenolol (100 mg/kg/day) proved to be active only during the period of treatment, whereas oxprenolol (100 mg/kg/day) was totally inactive. Propranolol and acebutolol were, however, inactive under identical conditions with SHR of the same strain but bred in another laboratory. The physiological difference between the two groups of SHR was confirmed by the fact that the resistance of their erythrocytes to osmotic shock was different. This suggests that all the beta-blockers do not necessarily have the same preventive activity with respect to hypertension in the SHR and that two groups of SHR bred in two different places can be physiologically dissimilar even though they originate from the same strain.

Human epidermal cells and squamous carcinoma cells synthesize a cytokine that augments natural killer cell activity.

Normal as well as transformed epidermal cells (EC) have recently been reported to produce a cytokine--EC-derived thymocyte-activating factor (ETAF), which according to its biologic as well as biochemical properties is indistinguishable from macrophage-derived interleukin 1 (IL 1). In the present study, the effect of supernatants (SN) derived from normal EC and a human squamous carcinoma cell (SCC) line were tested for their effects on natural killer (NK) cell activity. EC- as well as SCC-derived SN were able to augment in vitro NK cell activity of peripheral blood lymphocytes against K 562 cells. In contrast, adherent cell-derived, IL 1-containing SN did not affect NK cell activity. Upon high-pressure liquid chromatography (HPLC) gel filtration, ETAF and the EC-derived NK cell activity-augmenting factor (ENKAF) exhibited a similar m.w. However, by using reverse-phase HPLC, ETAF and ENKAF eluted as distinct peaks of activity, indicating that SCC cell-derived ENKAF is different from ETAF. Furthermore, ENKAF does not contain interleukin 2 (IL 2) or interferon (IFN) activity. The enhancement of NK cell activity was dose dependent and evident after 20 hr of preincubation of effector cells. Pretreatment of target cells with ENKAF did not affect the susceptibility of the target cells. The NK activity of large granular lymphocytes (LGL) purified by discontinuous Percoll gradient centrifugation and further depleted of high-affinity sheep erythrocyte rosetting cells was enhanced by ENKAF. In contrast, no NK cell activity was expressed by LGL-depleted T cell populations before or after treatment with ENKAF. In a single cell cytotoxicity assay in agarose, the number of lymphocyte binding to K 562 was not affected by ENKAF, but the frequency of dead conjugated target cells and presumably of active killer cells was increased by pretreatment with ENKAF. Additional incubation of LGL with ETAF did not further increase ENKAF-mediated augmentation of NK activity. In contrast to ETAF, ENKAF was not chemotactic for polymorphonuclear leukocytes. These results indicate that normal as well as transformed EC release a unique cytokine--ENKAF--which augments NK cell activity of LGL but is distinct from ETAF, IL 2, and IFN.