Thomas G. Pretlow

A MORE SENSITIVE AND SPECIFIC HISTOCHEMICAL PEROXIDASE STAIN FOR THE LOCALIZATION OF CELLULAR ANTIGEN BY THE ENZYME-ANTIBODY CONJUGATE METHOD

In an attempt to increase the sensitivity and specificity of immunohistochemical procedures using peroxidase-labeled antibody, we have modified the technique. This modification takes advantage of the pH optimum of horseradish peroxidase. The best buffer salts, hydrogen peroxide concentration, time of staining, fixation after incubation with labeled antibody and counterstains were determined empirically. Compounds similar to the substrate are known to undergo photooxidation, and specificity was enhanced by keeping the solutions in the dark.

DESTRUCTION OF ENDOGENOUS PEROXIDASE ACTIVITY IN ORDER TO LOCATE CELLULAR ANTIGENS BY PEROXIDASE-LABELED ANTIBODIES:

Convalent enzyme-antibody conjugates have been extensively employed for the immunochemical localization of cellular antigens (2. 5). This method is sometimes limited by the necessity to distinguish between endogenous enzyme activity and enzyme activity due to labeled antibody. This distinction is facilitated if the endogenous enzyme activity is destroyed while antigenic activity is preserved. Straus (6) used 1% sodium nitroferricyanide and 1% acetic acid in methanol as a fixative to destroy endogenous peroxidase activity. This fixative was recently applied in this laboratory to immunochemical localization of cellular antigens by a peroxidaseantibody conjugate. Streefkerk (7) destroyed endogenous peroxidase activity by treatment with methanol followed by hydrogen peroxide. Since methanol may destroy some cellular antigens, a different, methanolfree, fixing solution was sought. Figure 1 shows a 10-s frozen section of rabbit spleen which was fixed in absolute ethanol for 15 mm at room temperature. The section was incubated in a humidified chamber under a drop of 0.85% sodium chloride in 0.01 M sodium phosphate buffer, pH 6.8 (PBS), for 3 hr. The slide was then stained for peroxidase for 5 mm using a method similar to Graham and Karnovsky (3) as modified by Mazurkiewicz and Nakane (4). This slide shows the endogenous peroxidase activity of the rabbit spleen used. Figure 2 is the next serial section of the spleen. This section was fixed with 0.074% hydrochloric acid in ethanol (0.2 ml concentrated hydrochloric acid in 100 ml ethanol) for 15 mm at room temperature and incubated with PBS and stained as above. The endogenous peroxidase activity is abolished under these conditions. Figure 3 is the next serial section of the spleen. This section was fixed with 0.074% hydrochloric acid in ethanol for 15 mm at room temperature. The section was then incubated for 3 hr under a drop of PBS containing sheep antirabbit -y-globulin -peroxidase conjugate (0.50 mg protein/ml) prepared by a method similar to Avrameas (2). The section was washed twice for 1 mm and once for 30 mm in PBS and stained for peroxidase. In some instances, after immunohistochemical antigen location by peroxidase, the sections were counterstained with methyl greenpyronin (1) (Edward Gurr, Ltd., Michrome Labs, Bucks, England). The methyl green-pyronin did not affect the localization of the peroxidase reaction product and demonstrated that the cells which stained immunohistochemically were plasma cells. The antigenicity of the plasma cell -y-globulins was not destroyed by this fixation. By experiments similar to the above, plasma cells were also specifically stained in sections of rabbit appendix and lymph node using antirabbit ‘y-globulin prepared from both sheep and goat immune sera. Plasma cells were not stained by rabbit antiferritinperoxidase conjugates. The sections were fixed with 0.074% hydrochloric acid in ethanol as described. In these tissues, the endogenous peroxidase activity was destroyed while the antigenic activity remained intact. All slides were made in duplicate. In summary, new conditions for fixation are described which destroy endogenous peroxidase activity but not antigenic activity of rabbit -y-globulins. Hopefully, these conditions will be useful for other antigens as well.

Centrifugal elutriation (counterstreaming centrifugation) of cells.

Lindahl first described the separation of cells by velocity sedimentation utilizing a special technique (counterstreaming centrifugation) that was later modified slightly and renamed centrifugal elutriation. Centrifugal elutriation has been applied, with variable degrees of success, to the separation of hemopoietic cells, mouse tumor cells, testicular cells, and a variety of other specialized cells as well as cells in particular phases of the cell cycle. The capacity of the elutriator to separate large numbers of cells is its chief advantage. The purities of the separated cells have not been compared with the purities of cells separated by other methods in most cases; such comparisons would permit more sophisticated comparison of elutriation with other techniques for velocity sedimentation.

Pathogenesis of hodgkin's disease: Separation and culture of different kinds of cells from hodgkin's disease in a sterile isokinetic gradient of ficoll in tissue culture medium

The pathogenesis of Hodgkins disease is controversial. Some view the lymphocyte in Hodgkins tumor as an integral part of the malignant proliferation; others interpret the lymphocyte as a manifestation of the hosts attempt to reject the neoplasm. In an effort to study interactions between individual kinds of cells from Hodgkins disease, we have developed a method for disaggregating Hodgkins tumor using collagenase. The disaggregated cells have been frozen in a viable state and, after storage over liquid nitrogen, two kinds of cells have been obtained consistently in tissue culture from all five patients whose cells have been studied. Prior to culture, one of these cell types—a very homogeneous population of mature‐appearing lymphoid cells—has been separated from the other kinds of cells in 98.4 ± O.4% purity. This separation was accomplished using centrifugation in a gradient of Ficoll (polysucrose) in tissue culture medium. In culture, these cells divide and produce only lymphoid cells. The other kind of cell which can be cultured from Hodgkins disease—a histiocytic cell—can be widely separated from the lymphoid cells and does not give rise to lymphocytes in tissue culture. Using purified populations of the individual kinds of cells, it will be possible to study the possible cytotoxicity of the lymphoid cells for the histiocytic cells. To our knowledge, this is the first reported separation of lymphocytes from solid tumors, and the method may be broadly applicable in the investigation of lymphocytes which infiltrate solid tumors.

Cytologic Characterization of Pulmonary Alveolar Macrophages by Enzyme Histochemistry in Plastic

Quantitative morphometric analyses of macrophages should facilitate a more precise definition of the role of macrophages in neoplastic diseases and inflammatory diseases of lymphoid and hemopoietic tissues. The usefulness of the available phenotypic markers is affected by the extent to which they are expressed by macrophages in tissue sections. For our study of phenotypic markers for macrophages in human tissue sections, we selected lung, since pulmonary alveolar macrophages are more readily identified morphologically in tissue sections than macrophages in most other tissues. In 1-2 micron sections of freshly fixed lung embedded in methacrylate, 89.7% of pulmonary alveolar macrophages had histochemically demonstrable acid phosphatase; 86.2%, nonspecific esterase with naphthol ASD chloroacetate as the substrate; 86.2%, nonspecific esterase with alpha-naphthyl butyrate as the substrate; 80.5%, peroxidase; and 75.8%, iron. With respect to their expression of markers, the observed heterogeneity among pulmonary alveolar macrophages is interesting; this heterogeneity may reflect the degree to which they have been activated, the different periods of time since they arrived in the lung, and differences in their local environments. Except for peroxidase, all examined markers were as well demonstrated when tissues were fixed after storage over liquid nitrogen as when fixation was carried out with fresh tissue. Acid phosphatase and nonspecific esterase with the chloroactetate substrate gave bright colors that would facilitate morphometric analyses. The storage of tissue over liquid nitrogen will be equally satisfactory for the characterization of macrophages with histochemical markers and monoclonal antibodies.

Separation of hepatocytes from suspensions of mouse liver cells using programmed gradient sedimentation in gradients of ficoll in tissue culture medium

Abstract Liver cells were obtained in suspension using a solution of lysozyme in Jokliks modification of minimum essential medium. Hepatocytes were separated in 74.2 ± 12.9% purity from other liver cells having different densities using isopycnic centrifugation, in 97.1 ± 1.9% purity from other liver cells having different diameters using velocity or rate-zonal centrifugation. A previously reported computer integration of the differential sedimentation equation was employed in determining the gradient design and the speed and duration of centrifugation which would permit purification of hepatocytes from other liver cells. More than 98% of the hepatocytes separated by velocity sedimentation excluded trypan blue. Velocity sedimentation is superior to isopycnic centrifugation for the separation of hepatocytes from liver cell suspensions because it gives more highly purified hepatocytes and because it requires lower centrifugal forces for shorter periods of time.

CHANGES IN CYCLIN DEPENDENT KINASE INHIBITORS p21 AND p27 DURING THE CASTRATION INDUCED REGRESSION OF THE CWR22 MODEL OF PROSTATIC ADENOCARCINOMA

PURPOSE The expression of the cyclin dependent kinase inhibitors p21 and p27 was examined in prostatic adenocarcinomas following castration. MATERIALS AND METHODS Male nude mice inoculated with the androgen dependent human prostatic tumor CWR22 were castrated when the tumors reached a volume of 0.8 to 1.1 cm.3 and were sacrificed at 3, 7, 21, 28 and 42 days post-castration. An additional group of mice received a subcutaneous testosterone pellet at 21 days post-castration and was sacrificed at 28 days post-castration. The expression of the Ki-67 antigen, p21 and p27 was examined by immunohistochemistry. RESULTS The mitotic rate as well as the number of Ki-67 antigen positive cells decreased to 3% of intact control values by 7 days post-castration and were less than 0.01% of intact control values at 21, 28 and 42 days post-castration. The percentage of p21 expressing cells decreased from 15+/-2% in intact controls to less than 1% by 42 days post-castration. In contrast, the percentage of cells that expressed p27 increased from 25+/-3% in intact controls to 51+/-8% at 3 days post-castration and to 80 to 95% at days 7, 21, 28 and 42 days post-castration. Testosterone treatment from 21 to 28 days post-castration resulted in an increase in Ki-67 antigen positive cells to 200% of intact controls and a concomitant reduction in p27 expressing cells to about 50% of intact controls. Castration-induced changes in p27 expression were not observed in the CWR22R tumor, a transplantable relapsed derivative of the CWR22 tumor. CONCLUSION These findings suggest that p27 expression is regulated negatively by androgens and that increased expression of p27 in CWR22 xenografts may be involved in the suppression of proliferation following castration.

Partial purification of human colonic carcinoma cells by sedimentation.

We have purified epithelial cells from human colonic tumours by velocity sedimentation in an isokinetic density gradient of Ficoll in tissue culture medium. In frozen sections of colonic carcinoma, histochemically demonstrable N-acetyl-beta-D-glucosaminidase (HDAG) was observed primarily in epithelial cells. We used this enzyme as a histochemical marker of epithelial cells. Initial suspensions of cells from colonic tumours suspended with 0-25% trypsin contained an average of 24% of the nucleated cells with HDAG. In the purest fraction obtained from gradient centrifugations, an average of 74% of the nucleated cells contained HDAG. After centrifugation, the quarter of the density gradient which contained the most rapidly sedimenting cells was purified 2-4-fold over that in the initial suspension. Cells in this zone of the gradient also gave rise to colonies in soft agar. Cells from initial suspension resulted in 15-25% as many colonies of 7 or more cells in cultures inoculated with the same number of nucleated cells. For the most part, cells obtained from the other zones of the gradient did not give rise to colonies in soft agar.

Urinary hexosaminidase in patients with lung carcinoma

The specific activity of urinary hexosaminidase was determined in 58 patients with various histological types of lung carcinoma. Compared to an apparently healthy control population, 32/35 patients with widely disseminated disease showed elevated values and 18/23 patients with disease confined to the chest had normal hexosaminidase values. Detailed studies of 18 patients indicated that declining hexosaminidase values were associated with effective therapy, rising values generally accompanied progressive disease.

Acid phosphatase in prostatic tissue homogenates from patients with benign prostatic hyperplasia and prostatic carcinoma

Acid phosphatase activity biochemically in the primary tumor of 20 patients with prostatic carcinoma, was studied in an attempt to understand the basis for a correlation or lack of correlation between serum and/or bone marrow acid phosphatase levels and the presence and/or clinical behavior of prostatic carcinoma. The enzyme activity was similarly measured in 19 patients with benign prostatic hyperplasia as controls. On the average, enzyme activities were lower (P < 0.002) in the tissues from patients with carcinoma. There was no correlation of enzyme activity in tumor with the age of the patient, stage of disease, degree of differentiation of the tumor, or serum acid phosphatase activity. Cancer 52:155‐160, 1983. Cancer 52:155‐160, 1983.