The Journal of Experimental Medicine

Krupey, John; Gold, Phil; Freedman, Samuel O.

doi: 10.1084/jem.128.3.387pmid: 4299103

A procedure has been described for the purification of the carcinoembryonic antigens (CEA) of the human digestive system. Tumor tissue extraction in 0.6 M perchloric acid followed by paper block electrophoresis and column chromatography on Sephadex G-200 resulted in highly purified CEA preparations as determined by both immunological and physicochemical criteria. The properties and composition of five different purified CEA preparations derived from digestive system cancer metastases were examined. The findings demonstrated a high degree of uniformity amongst these samples. Sedimentation coefficients ranged from 6.9 to 8S. Each sample showed the presence of 14 different amino acid residues and six different carbohydrate constituents (four of which could be quantitated with the amount of material available for analyis). Studies of a purified CEA preparation from a primary hepatoma yielded results which, in some respects, differed from those obtained with the CEA samples of metastatic tumor origin. The implications of these variations were discussed with regard to the probable presence of non-CEA components in the hepatoma preparation. Of primary importance was the observation that the few normal adult digestive system tissues tested failed to show the presence of constituents similar to the CEA. This finding would seem to indicate that, in the adult, the carcinoembryonic antigens of the human digestive system are qualitatively tumor-specific and are not dectectable in comparable normal tissues. Footnotes Submitted: 12 March 1968

Medearis, Donald N.; Camitta, Bruce M.; Heath, Edward C.

doi: 10.1084/jem.128.3.399pmid: 4875325

Uridine diphosphate galactose 4-epimerase and phosphomannose isomerase-deficient mutants of Escherichia coli O111:B4 were studied to test the hypothesis that in E. coli a specific relationship exists between O antigenicity, virulence, and capacity to resist phagocytosis. The first mutant, designated J-5, produces a cell wall lipopolysaccharide, the side chains of which do not contain galactose, glucose, N -acetylglucosamine, or colitose. The second mutant produces a cell wall lipopolysaccharide which lacks only colitose. The capacity of these various organisms to kill mice was strikingly different. E. coli O111 was 1000 times as virulent as J-5, and 100 times as virulent as L-2. The capacity of the organisms to kill mice was correlated with their ability to resist phagocytosis and to persist in the peritoneal cavity. The parent strain of O111 resisted phagocytosis by macrophages in vivo and polymorphonuclear leukocytes in vitro. The mutants did not, and the organism most deficient in the saccharide component of its LPS was most susceptible to phagocytosis and least virulent. These results were corroborated by growing the mutants in appropriately supplemented media which permitted the synthesis of complete LPS, reversed the susceptibility to phagocytosis, and restored virulence. Finally, serological reactivity was consistent with previous observations which had demonstrated that the O antigenicity of E. coli is determined by the saccharide composition of its cell wall lipopolysaccharide. Despite the difference in the capacity of the various log-phase organisms to kill mice when injected intraperitoneally, purified lipopolysaccharides extracted from them did not differ significantly in their capacity to kill or produce fever. Thus virulence was shown to be independent of endotoxin activity which in turn seemed to be unrelated to the saccharide composition of the cell wall LPS. Collectively, these data provide at least a partial molecular definition of virulence in E. coli by demonstrating that the presence or absence of specific sugars in its cell wall lipopolysaccharide is a determinant of its antiphagocytic capacity and its virulence. Footnotes Submitted: 17 April 1968

van Furth, Ralph; Cohn, Zanvil A.

doi: 10.1084/jem.128.3.415pmid: 5666958

The origin and turnover of efferent populations of mouse mononuclear phagocytes has been described. Mononuclear phagocytes were defined as mononuclear cells which are able to adhere to glass and phagocytize. In vitro labeling studies with thymidine- 3 H showed that monocytes in the peripheral blood and peritoneal macrophages do not multiply and can be considered end cells in a normal, steady state situation. However, the mononuclear phagocytes of the bone marrow appear to be rapidly dividing cells. This conclusion was supported by in vivo labeling experiments. A peak of labeled mononuclear phagocytes of the bone marrow was found 24 hr after a pulse of thymidine- 3 H. This was followed, 24 hr later, by a peak of labeled monocytes in the peripheral blood. From these experiments it was concluded that the rapidly dividing mononuclear phagocytes of the bone marrow, called promonocytes, are the progenitor cells of the monocytes. Labeling studies after splenectomy and after X-irradiation excluded other organs as a major source of the monocytes. Peak labeling of both the blood monocyte and peritoneal macrophages occurred at the same time. A rapid entry of monocytes from the blood into the peritoneal cavity was observed, after a sterile inflammation was evoked by an injection of newborn calf serum. These data have led to the conclusion that monocytes give rise to peritoneal macrophages. No indications have been obtained that mononuclear phagocytes originate from lymphocytes. In the normal steady state the monocytes leave the circulation by a random process, with a half-time of 22 hr. The average blood transit time of the monocytes has been calculated to be 32 hr. The turnover rate of peritoneal macrophages was low and estimated at about 0.1% per hour. On the basis of these studies the life history of mouse mononuclear phagocytes was formulated to be: promonocytes in the bone marrow, → monocytes in the peripheral blood, → macrophages in the tissue. Footnotes Submitted: 8 May 1968

Shearer, G. M.; Cudkowicz, G.; Connell, Mary St. James; Priore, R. L.

doi: 10.1084/jem.128.3.437pmid: 5666959

Spleen cell suspensions of unprimed donor mice containing precursors of immunocytes have been transplanted into X-irradiated recipient mice. In the presence of antigen (sheep erythrocytes) these precursors, called antigen-sensitive units, gave rise to progeny cells secreting specific antibody. We studied quantitatively the production of cells releasing IgM hemolysins (direct plaque-forming cells), IgG hemolysins (indirect plaque-forming cells), and hemagglutinins (cluster-forming cells). We found that each of these immunocyte populations was distinct, i.e., that cells releasing agglutinins did not, as a rule, release hemolysins, and vice versa. We also found that cell populations secreting IgM hemolysins did not shift, under certain experimental conditions, to the production of IgG hemolysins during the primary immune response. By transplanting graded numbers of spleen cells, we succeeded in limiting to one or a few the number of antigen-sensitive units that reached the recipient spleen. We estimated thereby the frequency of antigen-sensitive units in donor cell suspensions and tested their potential for production of immunocytes of more than one type. Our results indicated that antigen-sensitive units were unipotent for they displayed in the spleens of unprimed donors the same restrictions of function and heterogeneity (antibody-specificity differentiation, antibody-class differentiation) found among antibody-forming cells. Furthermore, antigen-sensitive precursors for direct plaque-forming cells, indirect plaque-forming cells, and cluster-forming cells were detected in the spleens of unprimed mice in different frequencies, i.e., 1 in ∼ 10 6 , 1 in ∼ 7 x 10 6 , and 1 in ∼ 19 x 10 6 spleen cells, respectively. We concluded that relatively advanced differentiation of potentially competent cells occurs before sheep erythrocyte administration. The relevance of this finding for the broad spectrum of immunologic reactivities and for the heterogeneity of antibody responses to given antigens was discussed. Footnotes Submitted: 10 May 1968

Argyris, Bertie F.

doi: 10.1084/jem.128.3.459pmid: 5666960

1. Transplantation of peritoneal macrophages from thioglycollate-stimulated adult C3H donor mice, into 3-day-old C3H mice results in an enhanced antibody response to simultaneously injected SRBC. The increase in immuno-competence is even more pronounced when 1-day-old C3H mice are pretreated with adult macrophages and sensitized 3 days later with SRBC. Nonviable macrophages or nonviable spleen cells are ineffective. 2. There is a critical period in the development of the neonatal mouse during which the spleen cells benefit from the addition of adult macrophages. Treatment before or beyond this stage is ineffective. 3. Very high doses (20 million) of macrophages are less effective in stimulating antibody synthesis to SRBC than doses of 5 or 10 million, suggesting that a critical ratio of macrophages to immunocompetent cells may be required for enhancing antibody synthesis in young mice. 4. The results are discussed in the light of the hypothesis that newborn mice are immunologically deficient not because they lack immunocompetent cells but because they lack an antigen recognition or antigen-processing system in the form of functional macrophages. Footnotes Submitted: 13 May 1968

Åström, Karl E.; Webster, Henry de F.; Arnason, Barry G.

doi: 10.1084/jem.128.3.469pmid: 4875326

Experimental allergic neuritis (EAN) was produced in rats by the intradermal injection of an emulsion of peripheral nerve in Freund's adjuvant. Early lesions in perfused sciatic nerves were studied by phase, light, and electron microscopy at intervals up to 15 days following immunization. Circulating lymphocytes attached focally to the inner surface of blood vessels, primarily venules, to initiate parenchymal lesion formation. Attached cells had the hand mirror configuration typical of the motile lymphocyte. They subsequently flattened against the endothelial surface and then traversed the vascular wall by sinking into and passing through the cytoplasm of endothelial cells. The transgressor and transgressed cell membranes were intact and both cells retained their integrity. Lymphocytes began to transform and divide intravascularly; these events accelerated extravascularly. Although the migrating cells became larger and more pleomorphic in the perivascular regions, their essential character was in keeping with an origin from circulating lymphocytes. In many lesions, there was fluid with protein, possibly produced by the transformed extravascular cells. The described cellular events precede tissue damage and are likely instrumental in the myelin destruction which follows Footnotes Submitted: 15 May 1968

Van Winkle, M.; Levy, L.

doi: 10.1084/jem.128.3.497pmid: 5666961

In this report, the role of vascular allergy (i.e., hypersensitivity) in the potentiation of atherogenesis has been studied. In order to accomplish this, bovine serum albumin (BSA) was administered to rabbits in quantities sufficient to cause the occurrence of serum sickness (a type of hypersensitivity known to cause injury to the endothelial linings of certain blood vessels). This was immediately followed by the feeding of a special cholesterol-supplemented diet, which is known to be capable of initiating a high incidence of atheromatous disease in rabbits after prolonged feeding. Results indicated that those animals which received the combined treatment developed an incidence of pathology after only 2 wk of special diet which was not equaled in the diet-only control groups until they had been treated for 4 wk. This indicated that vascular allergy could potentiate lipemia-induced atherogenesis in the rabbit, and was in confirmation of an earlier study of a similar nature. Indeed, because of the relatively mild vascular injury caused by a single injection of BSA, it would seem as though vascular hypersensitivity was extremely effective in the potentiation of atherogenesis. In addition, these results may have given some indication of the degree of vascular injury necessary for the induction of irreversible atheromatous disease. While the incidence of lesions in serum sickness controls was seen to decrease with passage of time after BSA challenge, it appeared to increase after cessation of treatment in those animals which received the combined treatment of BSA plus 2 wk of cholesterol-supplemented diet. It would therefore appear that the atheromatous lesions seen as early as 2 wk after initiation of the experiment may already have been irreversible in terms of the resolution of established pathology. Footnotes Submitted: 3 May 1968

Cioli, D.; Baglioni, C.

doi: 10.1084/jem.128.3.517pmid: 5666962

Gel filtration analysis of the urinary proteins of some patients with myeloma has shown the presence of "fragments" of Bence Jones proteins which correspond to the variable half of these proteins. Experiments have been carried out to establish the origin of a "fragment" observed in a patient who excreted a large amount of this protein. Labeled homologous Bence Jones protein has been injected into this and other control patients. Excretion of labeled "fragment" has been observed in all. Analysis by peptide mapping and radio-autography of this labeled "fragment" isolated from the urine showed that the invariable half of the Bence Jones protein was not excreted; it seemed thus likely that the invariable half was metabolized to small peptides and free amino acids. A labeled Bence Jones protein which was excreted without any accompanying "fragment" was injected into the patient who excreted large amounts of "fragment." No excretion of labeled "fragment" was observed. It was thus concluded that the property of being degraded to "fragment" is characteristic of some "fragile" Bence Jones proteins and is not determined by the patient. Incubation with serum or urine of the "fragile" Bence Jones protein failed to produce any "fragment." "Fragments" of Bence Jones proteins are thus most likely formed during excretion of these proteins through the kidney and are products of the catabolism of Bence Jones proteins. Footnotes Submitted: 24 April 1968

Polley, Margaret J.; Müller-Eberhard, Hans J.

doi: 10.1084/jem.128.3.533pmid: 5666963

A method has been described for the purification and isolation of the second component of complement (C'2) from human serum. The protein is a ß 1 -globulin with an approximate molecular weight of 117,000. Immunochemical analysis using a variety of specific antisera, including a monospecific antiserum to the isolated protein, indicate that the C'2 protein represents a heretofore unrecognized human serum constituent. Isolated C'2 contained 2 x 10 9 "effective molecules" per microgram and 1000 hemolytically active C'2 molecules were required to produce a single hemolytically effective C'2 site on erythrocytes undergoing immune cytolysis. C'1 esterase treatment of C'2 resulted in reduction of both its electrophoretic mobility and its molecular size, the latter observation indicating fragmentation of the molecule. Direct evidence was presented for the physical presence of C'2 as an integral part of the enzyme C'3 convertase. Footnotes Submitted: 2 May 1968