Monoclonal Antikeratin Research Articles

Comparison of Keratin Monoclonal Antibodies MAK-6, AE1:AE3, and CAM-5.2

Two routinely used antikeratin monoclonal antibodies, AE1:AE3 (Hybritech Inc., La Jolla, CA) and CAM-5.2 (Becton-Dickinson, Mountain View, CA), were compared with a new antikeratin monoclonal antibody mixture, MAK-6 (Triton Biosciences, Inc., Alameda, CA). Various poorly differentiated epithelial neoplasms, lymphomas, melanomas, and sarcomas were studied with the use of the avidin-biotin-peroxidase technic. All three antibodies had similar staining profiles, however, some differences were noted. The MAK-6 and CAM-5.2 antibodies illustrated stronger staining for some grade III transitional cell carcinomas, whereas the AE1:AE3 was variably positive to negative in all cases. Three renal cell carcinomas were positive with all three antibodies, two were negative with all three, and one had scattered positive cells with MAK-6 only. All lymphomas and plasmacytomas were negative with MAK-6 and CAM-5.2, however, AE1:AE3 faintly stained two of three plasmacytomas and two of the seven large cell lymphomas. Two out of three hepatomas evaluated were strongly positive with CAM-5.2 and MAK-6 and variably positive with AE1:AE3. The third case had both positive and negative cells with all three antibodies. In conclusion, MAK-6 antikeratin antibody is as useful as AE1:AE3 and CAM-5.2 for identification of poorly differentiated epithelial neoplasms.

Nylon Brush Macular Amyloidosis

Long-term use of a nylon brush for back scratching by a 53-year-old white woman was associated with the development of typical macular amyloidosis. EKH4 monoclonal antikeratin antibody, which recognizes 50-kd neutral and acidic keratin species, labeled this amyloid. Confirmation of amyloid substance in the lesion included positive staining with Dylon and thioflavin T; immunohistochemical reactions with monoclonal and polyclonal antibodies against elastic fiber microfibrils (NKH1 and anti-P component), immunoglobulins (IgG, IgM, and IgA), and complement (C3); and electron microscopic identification of 6- to 10-nm straight filaments. Type IV collagen staining demonstrated a breakage and/or thickening of the dermoepidermal basement membrane above the amyloid deposition in the papillary dermis. Electron microscopic findings confirmed this phenomenon.

Local variations in the staining patterns of keratin antigens and blood group antigens in normal rodent oral epithelia

All rodent oral epithelia are orthokeratinized. However, morphological, immunohistochemical and biochemical studies have shown that regional differences exist. In the present study, intraregional variations in differentiation patterns of rat oral epithelia are demonstrated using monoclonal anti-keratin antibodies AE1 and AE2 and antibodies to blood group antigens B and H. Well-defined areas of rat buccal and hard palate epithelium differed from the general staining patterns of these epithelia. These areas were associated with a papillary surface contour. These local variations were not found in the strain of mice examined. The results suggest that physiologically different vertical compartments of keratinocytes exist within one and the same region of rat oral mucosa, a phenomenon previously recognized in detail only in the epithelium of dorsal tongue. The papillary structures may have some functional significance related to the processing of food similar to that suggested for lingual filiform papillae.

Antigenic phenotype of malignant mesotheliomas and pulmonary adenocarcinomas. An immunohistologic analysis demonstrating the value of leu M1 antigen

To evaluate the usefulness of an immunohistologic approach to the differential diagnosis of mesothelioma and pulmonary adenocarcinoma, the authors studied paraffin-embedded, fixed tissue sections from 50 primary adenocarcinomas of the lung and 28 mesotheliomas of the pleura by using a panel of monoclonal antikeratin, antihuman milk fat globule (HMFG-2), anti-Leu M1, and monoclonal anticarcinoembryonic antigen (CEA) antibody; we also used a conventional heterologous anti-CEA antiserum with and without prior absorption with spleen powder to remove antibodies to nonspecific cross-reacting antigen (NCA). Keratin was present in both mesotheliomas and adenocarcinomas and did not help in distinguishing between these two neoplasms. HMFG-2 was detected in 48 (96%), and Leu M1 was positive in 47 (94%) of the adenocarcinomas, but not in any of the mesotheliomas. By using conventional rabbit antiserum, the authors detected CEA in the majority of adenocarcinomas (96%), but also in two cases of mesothelioma. When the anti-CEA antiserum was absorbed with NCA, the number of positively reacting adenocarcinomas decreased considerably to 76%; however, after this treatment, none of the mesotheliomas gave positive reactions. The monoclonal anti-CEA antibody was reactive in 36 of the adenocarcinomas (72%), but in none of the mesotheliomas. Our results indicate that, in addition to HMFG-2 and CEA, the expression of Leu M1 antigen by most primary pulmonary adenocarcinoma (94%) and its absence in mesothelioma could be used as a valuable marker for primary adenocarcinoma of the lung that involves the pleura and permits its differentiation from mesothelioma.

Expression of Keratin Proteins in Deeply Invasive Basal and Squamous Cell Carcinoma: An Immunohistochemical Study

The expression of certain classes of keratin is associated with cell maturation and differentiation. During cell transformation and tumor development, the cell specificity of intermediate filament, keratin, is largely conserved. Taking advantage of this, we utilized monoclonal antikeratin immunohistochemical techniques to examine basal and squamous cell carcinomas as they became deeply invasive. Dyskeratotic keratinocytes and keratin pearls in squamous cell carcinomas (SCCs) stain with antikeratin antisera to larger keratins (65-67 Kd). At the deep tumor margins, SCCs no longer express larger keratins but retain expression of 50, 58 Kd, which are markers of keratinocytes derived from stratified squamous epithelial cells. This selective loss of expression of keratin polypeptide markers of differentiation in SCC is associated with progressively more aggressive biologic behavior as the tumor invades deeper structures such as muscle and bone. Recurrent basal cell carcinoma (BCC) which was of the nodular type when first excised, shows features of morphea-like BCC and of aggressive growth patterns at the deep invasive margin. At these deep margins, some tumors express markers of keratinization (65-67 Kd). While tumor cells retain the specificity of the intermediate filament, keratin, the individual cells express products of differentiation as measured by keratin expression independently of their cytologic atypia. At the deeper invasive margin of the tumor, the neoplastic cells synthesize keratin proteins in an unpredictable fashion.

Flow Cytometric Sorting and Keratin Analysis of Human Epidermal Basal Cells

AbstractA prerequisite condition for the elucidation of the expression of keratin subunits in human basal cells is obtaining highly purified basal cell fractions. A method has therefore been developed for the isolation of the basal cell sub‐population from normal human epidermis at very high purity. Epidermal cell suspensions obtained by trypsinization at 4°C were first enriched in basal cells by selective attachment to collagen. The cells were then stained in suspension with KLI monoclonal antikeratin antibody which had been shown to be reactive with suprabasal cells. The suprabasal cells contaminating the suspensions were then eliminated by selective flow cytometry sorting and basal keratinocyte populations of 99.5% purity were obtained. Analysis of the keratin composition of these cells showed that the large majority of basal cells contained two keratins, one from each keratin subfamily: type I acidic, 50 Kd [keratin no 14 as defined by Moll et al. (1)] and type II basic, 58 Kd [no 5]. Nevertheless, a small subpopulation of basal cells (2%) was shown to express, in addition to the 50/58 Kd subunits, the 56.5/65–67 Kd keratins (nos 10, 1–2) which are regarded as markers for cells committed to terminal differentiation.

Chronic sialadenitis

The cellular changes in salivary gland parenchyma with chronic inflammation were studied immunocytochemically with a panel of antibodies. Myoepithelial cells were labelled with antimyosin, duct cells with a polyclonal anti-callus prekeratin, a monoclonal anti-keratin CAM 5.2 and a monoclonal anti-keratin 7 (RPN 1162), and a subpopulation of basal duct cells with a monoclonal anti-keratin 16a. The wide range of changes observed were similar to those described following experimental duct ligation. One of the most striking features was the survival of myoepithelial cells surrounding persisting acini and ductal structures. Most of these ductal structures appeared to be either surviving intercalated ducts or were altered acinar cells. There was no evidence of myoepithelial or ductal hyperplasia. The 16a positive basally located duct cells which are conspicuous in normal glands, pleomorphic adenomas and in the epithelial islands in lymphoepithelial lesions (Palmer et al. 1985; 1986) were virtually absent, except in one specimen with mild inflammatory changes. If this cell type represents a reserve cell, then loss of it may preclude recovery of the remaining parenchyma following resolution of inflammation.

Basal cell tumour with eccrine differentiation (eccrine epithelioma)-a histochemical and immunocytochemical analysis of a case

Summary Basal cell epithelioma with eccrine differentiation is a tumour that has previously been characterized as a rare tumour in which the histological and histochemical differentiation towards eccrine epithelium has been taken to indicate a more aggressive clinical course. We present a further case of this tumour with studies of its pattern of keratin expression using a battery of anti-keratin monoclonal antibodies and conclude that the presence of simple epithelial markers within the tumour confirm the differentiation towards eccrine epithelium.

Flow Cytometric Sorting of Human Epidermal Pure Basal Cell Suspensions Using a Specific Antikeratin Monoclonal Antibody

A method is described for flow cytometric sorting of epidermal basal cells, based on the cellular keratin content. KL1, a monoclonal antikeratin antibody specific for the 56.5 kD polypeptide present in suprabasal cells was used to distinguish suprabasal from basal cells. After the action of Triton X-100, epidermal cells were stained in suspensions with KL1 antibody, and KL1-positive cells were separated by flow cytometry from KL1-negative cells (basal cells). This makes it possible to sort and analyze pure fractions of human epidermal basal cells since no less than 99% of the KL1 sorted cells were bearing bullous pemphigoid antigen. Therefore, the use of specific antibodies to cytoplasmic antigens can be of help in sorting pure fractions of cells of a particular tissue for further studies.

Distinct Keratin Patterns Demonstrated by Immunoperoxidase Staining of Adenocarcinomas, Carcinoids, and Mesotheliomas Using Polyclonal and Monoclonal Anti-Keratin Antibodies

The authors assessed whether distinct patterns for keratin could be demonstrated in 10 adenocarcinomas, 10 carcinoids, and 4 mesotheliomas by an immunoperoxidase reaction using 3 polyclonal and 3 monoclonal antibodies to keratin. When color development in diaminobenzidine (DAB) was allowed to proceed for less than 2 minutes, distinct patterns for keratin could be demonstrated using two polyclonal and two monoclonal antibodies; these were plasma membrane and/or web-like in the adenocarcinomas, punctate or crescentic in the carcinoids, and perinuclear in the mesotheliomas consisting of tumor cells with abundant cytoplasm. Immunoelectron microscopy using protein A colloidal gold confirmed these results. When color development in DAB was allowed to proceed for more than 2 minutes, only diffuse staining was seen. The distinct patterns of immunostaining for keratin obtained with the shorter color development were helpful in differentiating adenocarcinomas, carcinoids, and mesotheliomas.

Keratin species in type II pneumocytes in culture and during lung injury.

A detailed understanding of alveolar epithelial cell transitions during remodeling after lung injury requires the identification of specific markers. We have developed a panel of monoclonal antibodies against species of the intermediate filament protein, keratin. These individual species are recognized markers of the state of differentiation of various epithelial cells. These and complementary protein analytic methods have been applied to studies of isolated, enriched Type II pneumocyte preparations as well as to normal and injured lung tissues. Monoclonal antibody 24A3, initially raised against Morris hepatoma 7777 keratins, decorated a filament network in isolated cultured rat Type II pneumocytes by indirect immunofluorescence; it reacts by 2-dimensional polyacrylamide gel immunoblot procedures with an acidic, 46,000-dalton keratin. Monoclonal antikeratin antibodies AE1 and AE3, raised against human epidermal keratins, reacted poorly with isolated Type II cells; however, AE3 reacted by immunoblot technique with the 55,000-dalton keratin subclass. The bronchial epithelium reacted intensely with 24A3 as well as with a mix of AE1 plus AE3 in ethanol-fixed, paraffin-embedded sections of normal and injured rat lung. Alveolar regions of normal lung reacted poorly with all 3 antibodies, however, as visualized by light microscopy. At the same time, very large, presumptive epithelial cells in the alveolar regions stained intensely with 24A3 3 days after intratracheal instillation of bleomycin, whereas thin cells lining the alveoli in injured regions were intensely reactive 14 days after bleomycin treatment. These elongated cells may represent Type II pneumocytes in the process of converting to Type I cells.

Immunogold labeling of keratin filaments in normal human epidermal cells with two anti-keratin monoclonal antibodies.

We report on application of the highly sensitive and specific immunogold labeling method for ultrastructural investigation of keratin intermediate filament antigens in human epidermal cell suspensions. Triton X-100 pretreated cells proved accessible to the colloidal gold conjugate, thus enabling keratin filament bundles to be labeled. Anti-keratin KL1 and KL2 monoclonal antibodies were raised in mice after immunization with either human stratum corneum-isolated keratins or keratins extracted from human epidermal cells suspensions, respectively. Immunoelectron microscopy confirmed immunofluorescence and immunoperoxidase results of epidermal keratinocyte staining, and revealed two different antibody reactivity patterns: KL2 reacted with keratin filaments in keratinocytes of all epidermal layers, whereas antigen to KL1 was detected only on keratin of the suprabasal layers, not on the basal keratinocyte tonofilaments. The monoclonal antibody-recognized epitopes were specific for the keratin filaments. Vimentin-rich cells (melanocytes) were not stained in the same epidermal cell suspensions. Additionally, two distinct ultrastructural patterns of keratin filament epitope labeling were observed. KL1 and KL2 monoclonal antibodies react with two different antigenic determinants, depending on the stage of keratinocyte differentiation, and may therefore be used for immunohistochemical studies of various keratin-containing cells in normal and pathologic conditions.

Monoclonal antibody analysis of bovine epithelial keratins. Specific pairs as defined by coexpression.

We have characterized the keratin proteins of various bovine epithelial tissues by one- and two-dimensional gel electrophoresis, coupled with the immunoblot technique using AE1, AE2, AE3, AE5, CA20, BE14, and 6.11 monoclonal antikeratin antibodies. The results indicate that all known bovine keratins can be divided into two subfamilies. The "acidic" (Type I) subfamily consists of 41-, 43-, 45-, 46-, 50-, 54-, 56-, and 56.5-kDa keratins, all of which have a pI of less than 5.6, and most of them are recognized by our AE1 antibody, whereas the "neutral-to-basic" (Type II) subfamily consists of 55-, 57-, 58-, 62-65-, 66-, and 67-kDa keratins, all of which have a pI of greater than 6.0 and are recognized by our AE3 antibody. Tissue distribution data and cell culture studies show that, within the two subfamilies, keratins with similar "size ranks" form a "pair" as defined by frequent co-expression. Furthermore, within most "keratin pairs," the basic keratin is larger than the acidic one by 8-10 kDa. These results provide further support for the concepts of "keratin subfamilies" and keratin pairs and are consistent with the possibility that the acidic and basic members of at least some keratin pairs may interact specifically during in vivo tonofilament assembly and/or function. Immunoblotting data derived from the use of several monospecific antibodies show that although the size, charge, and pattern of expression of most bovine keratins are similar to those of the human counterparts, there are important exceptions to this rule.

Epithelial cell heterogeneity in the guinea pig thymus: Immunohistochemical characterization of four thymic epithelial subsets defined by monoclonal anti‐keratin antibodies

Keratins are a family of related polypeptides constitutive of the cytoskeleton of epithelial cells and are never found in nonepithelial tissues. Thymic epithelial cells (TEC), known to induce T cell differentiation, are the keratin-containing cells within the thymus. Using four monoclonal anti-keratin antibodies (KL1, KL4, AE2, AE3) directed against keratins of different molecular weight, we have investigated the guinea pig thymic epithelium. The immunohistochemical analysis of thymic cryostatic sections revealed that the keratin expression of TEC varied according to their location in the thymic lobula; the thymic cortex was specifically stained by AE3 whereas the thymic medulla and the subcapsular cortex were recognized by KL4. In addition, KL1 and AE2 exclusively labeled Hassall's corpuscles. The biochemical analysis of keratins extracted from the thymus showed that each TEC subset was characterized by an unique pattern of keratin polypeptides. This study extends the concept of thymic epithelium heterogeneity and suggests that anti-keratin antibodies which allow the typing of TEC subsets may be valuable tools for studying the differentiation of thymic epithelium and its in vitro function on T lymphocytes.

Coexpression of Neurofilament and Keratin Proteins in Cutaneous Neuroendocrince Carcinoma Cells

Four cases of neuroendocrine carcinomas (NECA) of the skin were studied by indirect immunofluorescence, using a monoclonal antikeratin antibody and a polyclonal antineurofilament antibody. Fifty to ninety percent and 80 to greater than 95% of the NECA cells stained with the antineurofilament antibody and the antikeratin antibody, respectively. Using double-labeling indirect immunofluorescence we could also demonstrate that, in 3 cases studied, some of the NECA cells, but not all, stained with both antikeratin and antineurofilament antibodies. These results, together with the recent knowledge of the intermediate filament protein type of normal Merkel cells (MC), tend to support the hypothesis that NECA cells do not originate from epithelial MC but from dermal neuroendocrine cells. A dual concept of intraepithelial MC and extraepithelial intradermal neuroendocrine cells, "from possible distinct origin," is proposed. Such a system has already been suggested for the neuroendocrine cells of the appendix and bronchial mucosae.

?Epimyoepithelial? islands in lymphoepithelial lesions

A panel of antibodies has been used in an immunoenzyme study in an attempt to characterize the cell types found in the 'epimyoepithelial' islands of lymphoepithelial lesions. Myoepithelial cells, which can be specifically stained with an anti-smooth muscle antibody were not found. The majority of cells stained with anti-prekeratin, suggesting a duct cell differentiation. A subpopulation of duct cells stainable with a monoclonal antikeratin (16a), previously described as occupying a basal location in normal salivary gland ducts, was clearly demonstrated. It is suggested that these cells may be important in the epithelial proliferation in these islands. The hyaline material within and surrounding the epithelial islands was positive for type IV collagen and therefore consists of basement membrane material.

Epidermoid metaplasias of xenotransplanted human tracheobronchial epithelium

Repopulation of rat tracheas of human tracheobronchial epithelial cells obtained from intermediate autopsies was achieved by introducing into de-epithelialized rat tracheas either pieces of donor tissue containing respiratory mucosa or epithelial cells produced by an in vitro amplification of these cells. After tracheas were sealed, and transplanted into the subcutaneous tissues of nude mice, a newly formed epithelium migrated over the denuded luminal surface. During this process, regenerative epidermoid metaplasias, consisting of the growth of thin stratified epithelium with keratinization but without atypia was observed. Four weeks after xenotransplantation, most of the luminal surface was covered by columnar epithelium with occasional patches of epidermoid metaplasia. When this epithelium was exposed to 7,12-dimethylbenzo[a]anthracene (DMBA) a thick epidermoid metaplasia with mild to moderate atypia was observed. This type of epithelium is seen one to three months after insertion of the DMBA-containing pellets into the tracheal lumen. Immunohistochemical staining with antikeratin monoclonal antibodies AE1 and AE3 revealed increased immunostaining in both regenerative and DMBA-induced metaplasias compared with that of untreated normal mucociliary epithelium. Although no differences between the two types of metaplasias were detected with AE1 and AE3, the use of involucrin immunostain showed important differences. Normal respiratory epithelium did not contain involucrin, but this protein was seen in the surface layer of regenerative epidermoid metaplasias. In DMBA-induced metaplasias, involucrin was found not only in the superficial cells but was also present in numerous suprabasal cells. The hyperplastic nature of these carcinogen-induced lesions, together with the presence of cellular atypia and an altered involucrin distribution pattern, suggest a preneoplastic state.

Monoclonal Anti-keratin (AE1) Reactivity in Routinely Processed Tissue from 166 Human Neoplasms

A large number of human neoplasms were tested for their keratin expression in routinely processed tissues by a simple, three-stage immunoperoxidase method using a broadly reactive monoclonal anti-keratin antibody AE1, which recognizes a number of keratin polypeptides distributed in a wide variety of epithelia. All carcinomas, with the exception of hepatocellular, adrenocortical, and basal cell carcinomas and occasional renal cell, pulmonary small-cell, and pulmonary large-cell anaplastic carcinomas, reacted with this antibody irrespective of differentiation, in most instances displaying staining of strong or moderate intensity in the majority of tumor cells. Equivocal results were obtained in some seminomas and dysgerminomas. Malignant melanoma, large-cell lymphoma, Hodgkin's disease, malignant histiocytosis, and stromal mesenchymal elements in all tumors did not show any reactivity with AE1. Even after routine processing, the determinant detected by AE1 is conserved and restricted to epithelial neoplasms. This suggests that AE1 would be valuable in the diagnostic distinction of anaplastic carcinoma from lymphoma and melanoma in routinely processed tissues.

Monoclonal antikeratin antibodies in the study of differentiation and malignancy in the human breast

Monoclonal antikeratin antibodies in the study of differentiation and malignancy in the human breast

Development, characterization and clinical applications of a new anti-keratin monoclonal antibody

Development, characterization and clinical applications of a new anti-keratin monoclonal antibody