Localization of cathepsin B in normal and hyperplastic human prostate by immunoperoxidase and protein A-gold techniques.

Cathepsin B, a lysosomal cysteine protease, was localized in normal prostate and benign prostatic hyperplasia (BPH) using immunoperoxidase and protein A-gold techniques. Our objective was to determine whether cathepsin B was involved in the prostatic epithelium affected by nodular hyperplasia. All samples were collected immediately after prostatectomy. Immunohistochemical studies showed that the enzyme was expressed in the supranuclear cytoplasm of columnar cells and in numerous basal cells of normal and BPH acini. The strongest localization of cathepsin B occurred in acinar basal cells; hence, it is possible that cathepsin B could be useful as a marker for such cellular elements. Stromal macrophages showed reaction products, but lymphocytes and neutrophils did not. In both normal and hyperplastic glands, the enzyme was localized by gold particles in lysosomes, secretory granules, and vacuoles of columnar epithelial acinar cells. Immunoelectron microscopic study also showed the presence of cathepsin B in the heterochromatin (condensed chromatin) and nuclear membranes of columnar and basal cells, but not in euchromatin or nucleoli. At present, the function of cathepsin B in the nuclei of basal and columnar cells remains unknown. However, the cathepsin B in the cytoplasmic compartment might be associated with the lysosomal function of the cells. The role of cathepsin B as a marker for basal cell participation in the development of prostatic lesions should be studied further.

Distribution and immunophenotype of the inflammatory cell population in the benign lymphoepithelial lesion (Mikulicz's disease).

Benign lymphoepithelial lesion (BLL) is an autoimmune process characterized by swelling and diffuse inflammation of the major salivary glands. Autoantibodies have been isolated from lymphocyte cultures obtained from affected salivary glands, but the pathogenesis is still unknown. Previous studies have shown that the predominant population of inflammatory cells is represented by helper T cells, with only brief mention of the B cell population. Twenty-five surgical specimens from patients with BLL were studied immunohistochemically. Antisera used included monoclonal antibodies LN-1 and LN-2 for B cells, LN-3 for cells expressing human leukocyte antigen-DR (HLA-DR) antigens, UCHL-1 for T cells, Leu-7 for natural killer (NK) cells, and T suppressor lymphocytes and the polyclonal antibody to S100 protein for dendritic cells. A peculiar distribution of the inflammatory infiltrate was observed in all cases, characterized by the presence of very irregular "germinal centers" with pseudopod-like extensions surrounding epimyoepithelial islands. Lymphoid cells in this location were reactive with LN-1 and LN-2 antibodies. These structures were surrounded by a "mantle" of mixed small B and T lymphocytes. A well-defined "interfollicular" zone was composed of cells strongly reactive with UCHL-1 and LN-3 antibodies, indicating the presence and activation of T cells. Dendritic cells defined by S100 and LN-2 reactivity were intermixed with epimyoepithelial cells, and were identified in 18 cases. Epithelial expression of HLA-DR antigens was restricted to inflamed areas. In contrast to previous reports denying the presence of Leu-7-positive cells in these lesions, cells reactive for this antibody were identified in 13 of 20 cases, predominantly within germinal centers. The presence of dendritic cells, complex organization of the inflammatory infiltrate into well-defined B cell proliferation centers and activated interfollicular T areas, and the abnormal expression of HLA-DR antigens in epithelial cells support an antibody-mediated destruction of the epithelial cells in this disease.

The use of proteolysis with ficin, for immunostaining of paraffin sections. A study of lymphoid, mesenchymal, and epithelial determinants in human tissues.

In order to maximize staining, modifications of immunostaining methods have included proteolytic enzyme digestion of tissue. The authors performed a study of the effect of ficin in 110 paraffinized specimens, including tonsil, lymph nodes, benign vascular and nerve sheath tumors, and various carcinomas and sarcomas. This agent was compared with pepsin and bromelain, as alternative proteases. A panel of monoclonal and polyclonal antibodies was used, with and without previous digestion by ficin, pepsin, and bromelain. A score was assigned to each stain, based on the number and intensity of reactive cells. Ficin enhanced staining markedly in immunostains with antibodies to keratin and Factor VIII-related antigen (F8RAG). Conversely, it abolished staining for LN-2 (a lymphoid marker) and weakened reactivity for S-100 in nerve sheath tumors. Bromelain produced similar results, except that it enhanced S-100. Pepsin was comparatively less active than ficin and bromelain overall but did produce the greatest amplification of vimentin staining in sarcomas. Digestion with any of the three enzymes failed to influence reactivities of leukocyte common antigen, UCHL-1 (a lymphoid marker), alpha-1-antichymotrypsin, carcinoembryonic antigen, epithelial membrane antigen, and blood group isoantigens. These results may reflect a dissimilar recognition of peptide targets in some antigenic proteins, by ficin, bromelain, and pepsin. Hence, one enzymatic agent is unlikely to produce optimal staining for all determinants. With this proviso, however, ficin appeared to be the best general enhancer for antigens known to require vigorous digestion (e.g., keratin; F8RAG) for optimal reactivity in paraffin sections.

Avidin-biotin-peroxidase-antiperoxidase (ABPAP) complex. An immunocytochemical method with enhanced sensitivity.

The avidin-biotin-peroxidase-antiperoxidase complex method (ABPAP) of immunohistochemistry employs the sequential application of the peroxidase-antiperoxidase complex (PAP) and the avidin-biotin-peroxidase complex (ABC) technics. To assess the efficacy of ABPAP in the detection of cellular antigens and to compare its utility with that of protease digestion technics, the authors studied cytokeratin (CK) reactivity in six examples each of colon, prostate, and breast cancer and Leu-M1 reactivity in five ductal breast carcinomas. ABC, PAP and ABPAP were applied to these cases alone and in combination with prior pepsin digestion of deparaffinized sections. With respect to the number of cells that stained and their intensity in each case, there was significant enhancement of CK and Leu-M1 reactivity with ABPAP, as compared with ABC and PAP when pepsin digestion was not employed. Further, ABPAP provided undigested CK staining results comparable to those seen with the use of ABC or PAP with pepsin digestion. Protease treatment was uniformly detrimental to the visualization of Leu-M1, regardless of the method employed. In summary, ABPAP was clearly superior to ABC and PAP in all settings analyzed. It may prove to be useful adjunct in the immunohistochemical visualization of tissue antigens, when more routine technics yield suboptimal results.

Three immunostaining techniques for the localization of leukocyte common antigen in formalin-fixed, paraffin-embedded dermatological biopsy specimens.

Although leukocyte common antigen is a valuable discriminant between lymphoreticular proliferations and other histologically similar neoplasms, several publications have described difficulties in adequately labeling this determinant in formalin-fixed, paraffin-embedded tissues. We studied 662 paraffinized cutaneous and noncutaneous neoplasms for the presence of leukocyte common antigen, using an extended avidin-biotin-peroxidase complex (ABC) method, a combined ABC-peroxidase-antiperoxidase technique, and a biotin-strept-avidin procedure. Each of the three immunohistochemical techniques produced effective results in the detection of leukocyte common antigen in all malignant lymphomas, but none of them produced false-positive staining in nonhematopoietic lesions. These results suggest that antibodies to leukocyte common antigen are highly specific and that the methods employed in this study allow for excellent diagnostic sensitivity in the recognition of cutaneous lymphomas.

Analysis of fixation effects on immunohistochemical localization of prostatic specific antigen in human prostate.

We studied human prostatic specific antigen (PSA) localization in human prostate to investigate the possibility that the previously reported variations in the intensity of antigen staining were due to fixation and embedding methods. We have evaluated the effects of physical and several chemical fixatives on prostate samples obtained immediately after prostatectomies and radical cystectomies. Our analysis of fixation effects and immunohistochemical staining of polyclonal antibody to PSA indicates that the formalin fixation and paraffin embedding methods used previously did provide optimum localization of the antigen and the variations in the intensity of PSA staining could not be attributed to the methodology. Although PSA staining was relatively uniform in the lower grade neoplastic tumors, the higher grade, moderately to poorly differentiated tumors showed intense-through-weak PSA localization or no PSA staining suggesting that PSA staining intensity was not uniformly related to tumor differentiation.