Factor XIII is a blood protransglutaminase that is distributed in plasma and platelets. The extracellular and intracellular zymogenic forms differ in that the plasma zymogen contains A and B subunits, while the platelet zymogen has A subunits only. Both zymogens form the same enzyme. Erythrocytes, in contrast, contain a tissue transglutaminase that is distinct from Factor XIII. In this study other bone marrow-derived cells were examined for transglutaminase activity. Criteria that were used to differentiate Factor XIII proteins from erythrocyte transglutaminase included: (a) immunochemical and immunohistochemical identification with monospecific polyclonal and monoclonal antibodies to Factor XIII proteins, (b) requirement for thrombin cleavage to express activity, (c) pattern of fibrin cross-linking catalyzed by the enzyme, and (d) different electrophoretic mobilities in nondenaturing gel systems. By these criteria human peripheral blood monocytes, peritoneal macrophages, and monocytes maintained in culture contain an intracellular protransglutaminase that is the same as platelet Factor XIII. The monocyte-macrophage protein is thrombin-sensitive, and under appropriate conditions there is no enzyme expression without activation of the zymogen. Both the monocyte-macrophage zymogen and enzyme have the same electrophoretic mobilities as platelet Factor XIII zymogen and enzyme. Antibody to A protein reacts with the monocyte-macrophage protein. B protein is not associated with this intracellular zymogen. By immunoperoxidase staining monocyte-macrophage protein seems to be localized in the cytoplasm, similar to the known cytoplasmic distribution of platelet and megakaryocyte Factor XIII. These procedures were also used to study populations of human granulocytes and lymphocytes, and protransglutaminase activity was not observed in these cells.

Malignant effusions and tumour tissue obtained at surgery provided material for a study of the prognostic value of the various inflammatory cells in the prognosis of human ovarian cancer. Ascitic fluids predominantly contained inflammatory cells; tumour cells, both singly and in clusters, were a minor component. Tumour cells were usually in excess in dispersed solid material. Some patients had significant proportions of lymphocytes and macrophages in their solid tumour, and these patients invariably responded to therapy. Sedimentation-velocity separation at unit gravity provided tow populations of inflammatory cells. One consisted of mononuclear cells similar in size to those in the patients blood: the other consisted of one or more large macrophage populations, distinct in morphology and enzymatic markers from both blood monocytes and each other. T lymphocytes were enriched in ascites fractions (78%) and in the tumour-derived mononuclear fraction (71%) compared to patient blood (60%). The T-cell subset characterized by ANAE reactivity was markedly depleted in the tumour-infiltrating fraction (17%) compared to patient blood (62%) or patient blood (51%). Esterase-positive monocyte-like cells were more frequent in the tumour-infiltrating fraction (17%) than ascites (7%) or blood (12%). B lymphocytes were infrequent in solid tumours and difficult to assess in ascites. Histiocyte-like macrophages were present in the higher-velocity tumour-cell containing fractions of both solid and ascitic material. The variation in infiltrating cells between patients and between tumour and ascites of the same individual was marked.

Summary.-Macrophages have been isolated from ascitic and collagenase -dispersed tumours from patients undergoing surgery for ovarian cancer. Macrophages were present in varying proportions in both sites, though the ratio of macrophages to tumour cells was higher in ascites. Marked variation in size (as detected by sedimentation velocity) and cytochemical markers in the macrophages was noted. Highly enriched macrophage fractions were isolated from the ascites and collagenasedispersed solid tumours by a combination of sedimentation velocity and selective EA RFC or adherence techniques. Suppressor activity in the PHA assay was detected in tumour macrophages (4/10 giving>50% inhibition), ascitic macrophages (1/15) and blood monocytes (2/7). Lymphocyte fractions from tumours were unresponsive to PHA and failed to suppress the blood response. Suppressor activity was also present in the purified tumour-cell fraction of 6/14 patients.ADCC activity was tested in a few patients. When the activity was determined against the SB target cells, tumour-derived macrophages were inactive, whereas the ascitic fraction showed low but significant activity which averaged much lower than patient blood values. The ADCC assays carried out with the CRC target cell indicated activity within the range of patient blood values in 4/4 ascites and 2/4 tumour macrophage fractions.Cytotoxicity was also assessed against co-purified autologous tumour cells. Although activity was detected in many of the tests, the results seemed to reflect target cell sensitivity. There appeared to be a correlation between cytotoxicity with test macrophages and normal blood mononuclear cells.The results indicate that the cytochemical heterogeneity and the variation in size between macrophage fractions is associated with a spectrum of activities.WE HAVE INITIATED a study of immune competence of the various inflammatory cell types infiltrating primary solid and ascitic ovarian tumours. In the first paper (Haskill et al., 1982a) we outlined the methods used to isolate these cells and characterized the cell markers associated with infiltrating cells from these tumours. Two classes were characterized; 1 sedimented at < 6 mm/h and was similar in size to most blood mononuclear cells; the other was composed of larger, strongly adherent macrophages distinct from blood monocytes, which sedimented with the tumour cells. In the second communication (Haskill et al. 1982b) we investigated effector-cell functions (PHA, ADCC and NK) associated with blood and bloodequivalent inflammatory cells present in both ascitic and solid tumours. The data indicated that all tumour-derived effector-cell tests were markedly depres-

Summary.-Mononuclear cell fractions were isolated from blood, ascites and solid tumours of patients undergoing surgery for Stages III and IV PHA responses of patient blood and ascites fractions were about half that of normal blood. Tumour-infiltrating lymphocytes (TIL) were less than 10% as responsive as normal blood. The depressed PHA responses of the TIL were not due to the presence of a suppressor cell population. NK activity of patient blood was less than that of normal blood, but not as much as the ascites or TIL cells. The activity of the ascites-derived lymphocytes was enhanced by treatment with interferon. ADCC activity against both CRBC and SB cells was normal or higher than controls in patient blood, and depressed in the ascites-derived fractions. TIL responded to <10% of the patient blood values.The results indicate a lack of response by ascitic and TIL cells in assays dependent on FcR -bearing effector cells and a greater loss of PHA-reactive cells from the tumour than from blood and ascites. These data could result from intratumour inactivation, or a failure of the particular subset to localize either In the ascites or the tumour site.

Presumptive sarcoma cells have been isolated from primary MSV tumors induced in normal, immunosuppressed (ALG) and athymic nude mice. These cells were atypical in appearance, induced tumors in secondary hosts and expressed the viral antigens gp70, p30 and p12. In vitro growth characteristics of the MSV cells were tested in a variety of assays defining cell transformation. None of these indicated that the virus-positive cells were transformed. Sex chromosome marker studies were carried out to determine if secondary tumor arose solely from proliferation of donor cells or through infection of host tissue. Tumors from female irradiated mice induced by injections of tumor cells derived from male mice contained a high proportion of metaphases of non-donor origin, indicating that infection is an important but not the sole mechanism involved in tumor transfer. These data also demonstrated that many of the sarcoma cells are damaged as a result of infection as shown by the presence of chromosome breaks and aneuploid metaphases. Histologic sections of both regressor and progressor tumors showed that the virus-positive cells were scattered as single cells surrounded by inflammatory cells, rather than as masses of dividing virus-positive cells found in the MSV-induced transplanted tumor. Collectively, these data support the contention that the MSV tumor is the manifestation of a response to a highly noxious virus infection rather than a tumor of dividing, transformed malignant cells.