Afp specific fluorescence is present in the epithelium of the external layer and in the mesenchyme of embryonal type inside the cyst. E-h: sections of a small granule consisting of a central core of embryonal carcinoma cells and a peripheral epithelial layer, g, h: higher magnification of region shown. G, h: area analogous. Magnifications: a, b, e, f: x28; c, d, g, h: x112. Mesenchymal elements, especially in medium-sized and large cysts, exhibited, as a rule, very bright afp-specific fluorescence. Embryonal carcinoma cells usually did not contain afp, but in two cysts bright fluorescence was observed in the inner cell mass after treatment with anti-afp, in the absence. It could be supposed that in cysts, unless they contained large quantities of γg, the test for the latter protein was not an adequate control to decide on passive accumulation of afp. On the contrary, high afp concentration in cyst medium could be the cause of its detection in mesenchymal and other internal structures of cysts.
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Under the reporting epithelial layer there was mesenchyme of the embryonal type, sometimes forming structures like primary blood vessels, as in normal yolk sac and agglomerations of embryonal carcinoma cells. In small cysts and granules fewer cavities could be found. Sometimes they contained only a central core of embryonal carcinoma cells and a peripheral layer of epithelium resembling visceral yolk sac. In other cases inside these bodies mesodermal and ectodermal elements could be also found. Γg was present in afp-containing cysts in insignificant amounts, on the surface of the epithelial layer, less frequently inside its individual cells, and, in trace quantities, in mesenchymal elements of the inner cavity. The degree of afp-specific fluorescence in cysts was, as a whole, higher than in sections of solid tumors. The protein was regularly found in the cytoplasm of the outer layer of cysts. It should be noted, however, that afp was usually not observed in all the cells of this layer, sometimes being evident only in a small proportion of cells and completely absent from the rest of them. High resolution click here figure 4 afp and γg in the sections of medium-sized cyst and a granule from the intraperitoneal ascites, a; c; e; g: incubation with anti-afp-antibodies; b; f: incubation with anti-γg-antibodies; a-d; sections of a medium-sized cyst; c: higher magnification of the. D: region analogous to that shown.
Luminescence of a still lower degree was found also in squamous epithelium arranged in keratin pearls. (1960) supposed that such structures could possibly be derivatives of the visceral yolk sac. High resolution click here figure 3 Unidentified afp-containing structures in the sections of subcutaneous teratocarcinoma ott 6050. A, c, e: incubation with anti-afp-antibodies; f: incubation with anti-γg-antibodies; b, d, g: areas shown in a, c and e after hematoxylin-eosin essay staining. Afp in cysts and granules developing after intraperitoneal inoculation of teratocarcinoma In four cases, when cysts were found in the ascites, afp titers in the animal sera and ascitic fluids were 1:32, 1:8, 1:4 and 1:2, the difference being correlated to the number of cysts. Morphologically, cysts were very similar to those described by pierce. (1959, 1960 and their external epithelium was indeed very much like that of the visceral yolk sac (Fig. Large and medium cysts with a diameter of 2-5 mm presented hollow transparent vesicles.
Note absence of afp-specific fluorescence in the epithelium of the intestinal type, e, g: regions of the section corresponding to c and f after hema-toxylin/eosin staining. Magnifications: a, b: x28; c, d, e, f, g: x112. High resolution click here figure 2 afp in the epithelium of two small tubes in the section of subcutaneous teratocarcinoma ott 6050. A, c, e: incubation with anti-afp antibodies; b: incubation with anti γg antibodies; c and e are higher magnifications of areas shown. Note more intensive afp-specific fluorescence in the apex of the investing cells, d and f: regions analogous to c and e after hematoxylin-eosin staining. Magnifications: a, b: x28; c, d, e, f: x112. The intensity of fluorescence in such afp-containing structures varied from very bright to almost background, and afp was frequently detected in only a small proportion biography of cells of the lining. Besides, sufficiently bright afp fluorescence was episodically found in epithelial sheets, cell groups, and individual cells the origin of which we could not identify, although all of them were relatively well differentiated (Fig.
In the section presented in Figure 1 there was no afp in adjacent epithelium which was more intestinal in type (Fig. In the same section afp was present in several tubes and rosettes formed by cells which resembled those of the former afp-positive epithelium. In many cases afp-positive epithelium had no similarity with the epithelium of the visceral yolk sac (Fig. Usually afp-specific fluorescence was seen in the whole cytoplasm of cubic epithelium while in more differentiated prismatic epithelium it was restricted to the apex of the cells. High resolution click here figure 1 afp and γg in the consecutive sections of solid teratocarcinoma ott 6050 containing epithelium resembling that of the visceral yolk sac. A, c, f: incubation with anti-afp antibodies; b, d: incubation with anti- γg antibodies; c and d are higher magnifications of areas shown in a and. Note the absence of γg in the structures containing afp and vice versa, f, g: region of the same cyst as in Fig.
Protein, synthesis, take place?
The diluted media from large cysts and corresponding ascitic fluid diluted 1:40 were essay separated in the same run for comparison. Electrophoretic fractions eluted after separation from different regions of the column were identified by agar precipitation using standard test-systems for mouse afp, albumin, transferrin and γg (Goussev and yazova, 1970). Afp concentration afp concentration in sera and ascitic fluids of mice bearing teratocarcinomas was determined using the standard agar precipitation procedure with the test-system (Abelev and bakirov 1967). Results afp in solid teratocarcinomas The afp level in the serum of mice bearing teratocarcinoma was always low; serum titer with standard test-system for afp never exceeded 1:2, and sometimes only a slight curvature of the precipitin line of the test-system was observed in agar. With the immunofluorescence technique afp was not revealed in the main area of the tumor sections. It was lacking in embryonal carcinoma, nervous, muscle, cartilage and connective tissues even in those cases when in these elements bright fluorescence specific for γg was observed.
The latter protein served as a reference for non-physiological serum presence (see engelhardt., 1971). Moreover, if several specimens of one tumor were taken, afp was sometimes found in the sections of only a few of them, for example in one of three specimens taken from the first generation, in one of two specimens of the fourth generation, etc. Afp-containing structures usually occupied an insignificant portion of the sections and were clearly seen contrasting with dark background even when afp level in the serum was quite low. Fluorescence in these areas was completely absent in consecutive serial sections incubated with anti-afp antibodies neutralized with purified afp preparation. Γg was usually not observed in such structures. Most intensive and frequent afp-specific fluorescence was observed in the cytoplasm of epithelial cells of endodermal origin, lining cysts and tubes of various dimensions. Sometimes afp-containing cells resembled those of visceral yolk sac as described by pierce., 1959, 1960 (see rugh, 1968, fig.
Cells of the external layer covering the cysts and granules very closely resembled those of the visceral yolk sac. We exploited this route of inoculation in order to increase the number of such cells in the tumor and study their relation to afp synthesis in teratocarcinomas. In four cases when cysts and granules were present they were collected from the ascites, washed in Petri dishes in several changes of large volumes of Ringer solution, fixed, dehydrated, and embedded in paraffin with wax by the same procedures as tumor specimens. Medium from the cysts, larger cysts (diameter 3-5 mm) were placed after washing onto a dry region of a petri dish and punctured with thin glass capillaries. The internal liquid was collected and diluted.25 ml of Tris-HCl buffer,.7 (1:20-l:40 centrifuged for 15 min at 13,000 rpm and subjected to analytical polyacrylamide gel disc-electrophoresis.
Antibodies against afp, antibodies were prepared and checked for monospecificity as described previously (Engelhardt., 1971). They reacted only with afp in agar precipitation and were completely neutralized in immunofluorescence after addition of a slight excess of purified murine afp. These antibodies were as described in a previous publication (Engelhardt., 1971). Immunomorphological localization, this was performed on serial paraffin sections of tissues fixed with ethanol-acetic acid. Treatment of tissue specimens, as well as the technique of immunofluorescence, was described in detail earlier (Engelhardt., 1971). Histological examination, for histological examination, we used either the sections employed for immunofluorescence localization afp after disembedding and staining with hematoxylin-eosin, or consecutive serial sections. Analytical polyacrilamide gel disc-electrophoresis, this was performed according to davis (1964) in 4 and 7 gels with slight modifications (Goussev, 1969; goussev and yazova, 1970).
Protein, synthesis, essay research
Transplantable teratocarcinoma o tt 6050, this neoplasm was also provided. The initial tumor was derived from a 6-day embryo grafted to the testis of an adult mouse in 1967 and subsequently transplanted subcutaneously in males by tissue suspension in saline at 25- to 35-day intervals. Five consecutive generations were examined during this study. In all cases the tumor sections besides the embryonal carcinoma contained different tissues. Most prominent was nervous tissue with pathological nervous tubules. Also present in these sections were muscle tissue sometimes resembling the heart paperwork muscle, cartilage, bone, numerous glands, tubes and cysts lined with different types of epithelium, resembling that of intestine, or visceral yolk sac, or ectodermal epithelium with keratinization, bronchogenic or squamous epithelium. Free-floating bodies were obtained in a group of animals according to pierce. These authors found that mice of the 129th strain, if short injected intraperitoneally with a suspension of subcutaneous teratocarcinoma, develop solid tumors, hemorrhagic ascites, and, in the majority of cases, numerous cysts and small granules floating in the ascitic fluid.
In the metastasis to a lymph node characterized as embryonal epithelioma, afp was found in some of the cells distributed in groups or trabecules. No further characterization of afp-containing structures was reported. In the present study localization of afp has been investigated in transplantable murine teratocarcinomas. This experimental model is very similar to human primary teratocarcinomas (Stevens and Hummel, 1957; Stevens, 1958). Materials and Methods, mice, a special subline of inbred strain business 129, designated 129/sv-sl cp, was used. These mice were kindly provided. Stevens of Jackson Laboratory, bar Harbor, maine, usa.
the one hand provides for tumor growth and on the other hand differentiates into the somatic tissues of teratocarcinomas (Pierce., 1959, 1960; Kleinsmith and pierce, 1964). Cells analogous to hepatocytes are very rarely found in human teratocarcinomas, and in those of mice they have not been described at all (Stevens and Hummel, 1957). It has been suggested, therefore, that afp synthesis in teratocarcinomas is carried out by structures analogous to yolk sac, which are rather common in these tumors (Abelev, 1971; Gitlin., 1972). Other authors (Kahan and levine, 1971) considered the non-differentiated cells of embryonal carcinoma as a possible site of afp synthesis. The immunofluorescence technique seemed to be a useful approach to estimate the cells connected with the afp-production. The only previous report about afp localization in teratocarcinomas is that of Mawas. In the frozen sections of human teratocarcinoma bright afp-specific fluorescence was observed in the cytoplasm of pseudostratified epithelium lining a cyst, and faint fluorescence was present in the solid zones of the tumor.
By analytical disc-electrophoresis in polyacrylamide gel, cysts were found to contain albumin, afp, transferrin and gamma-globulin, the relative proportion of afp being considerably higher than in the ascitic fluid as compared with the other proteins mentioned. It is suggested that like afp localization in teratocarcinomas corresponds to the sites of its synthesis in the embryo and that afp synthesis is a specific function of certain forms of endodermal differentiation. It is well established that two forms of tumors hepatocellular cancer and teratocarcinomas of animals and human beings are regularly accompanied by reappearance in the serum of embryo-specific component α-fetoprotein (AFP). Afp is the main component of embryonal sera of various mammalian species. It can easily be distinguished from other fetal serum proteins by its physico-chemical and especially immunological properties: afp of different species share common antigenic determinants (Abelev, 1971). During normal ontogenesis afp is synthetized by the yolk sac and liver of fetuses (Gitlin and boesman, 1966; Abelev, 1965; Wise and Oliver, 1966; Abelev and bakirov, 1967; Abelev, 1971). In the tumor-bearing animals and in cancer patients the afp synthesis is effected by tumor cells proper (Abelev., 1963; Kahan and levine, 1971; Abelev, 1971). However, the cellular basis of afp production in tumors is not clear because of insufficient direct experimental data. What cells are responsible for afp synthesis in teratocarcinomas?
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Cancer: 11, 448-459 (1973) by,. Tumor Immunochemistry and diagnosis Laboratory,. Epidemiology and Microbiology, ussr academy of Medical Sciences, moscow, ussr. Received: September 18, 1972, and in revised form December 4, 1972. Embryo-specific serum component α-fetoprotein (AFP) was localized by the indirect eksempel immunofluorescent technique on paraffin sections of transplantable teratocarcinoma of strain 129/sv-si cp mice. Afp was found in several types of structures comprising an insignificant part of the total section area. Most frequent and bright afp-specific fluorescence occurred in the epithelium of endodermal origin lining cysts and tubules; in addition, afp was found in sheets of cells, groups or individual cells of non-identified origin and in squamous epithelium arranged in pearls. In cysts and granules isolated from the ascitic fluid, developing after an intraperitoneal injection of teratocarcinoma suspension, afp was found in cells of the external layer, very similar to those of the visceral yolk sac, and in the embryotype mesenchyme inside the cysts.