ABSTRACT
This study addresses the issue of the effect of immunomodulating therapies in the target organ-the central nervous system (CNS)-in the case of multiple sclerosis. Copolymer 1 (Cop 1, Copaxone, glatiramer acetate), an approved drug for the treatment of multiple sclerosis, is a potent inducer of Th2 regulatory cells in both mice and humans. Highly reactive Cop 1-specific T cell lines that secrete IL-4, IL-5, IL-6, IL-10, and transforming growth factor-beta in response to Cop 1 and crossreact with myelin basic protein (MBP) at the level of Th2 cytokine secretion were established from both brains and spinal cords of Cop 1-treated mice. In contrast, no reactivity to the control antigen lysozyme could be obtained in lymphocytes isolated from CNS of mice injected with lysozyme. Adoptively transferred labeled Cop 1-specific suppressor cells were found in brain sections 7 and 10 days after their injection to the periphery, whereas lysozyme-specific cells were absent in the CNS. Hence, Cop 1-induced Th2 cells cross the blood-brain barrier and accumulate in the CNS, where they can be stimulated in situ by MBP and thereby exert therapeutic effects in the diseased organ. This therapeutic effect was manifested, in brains of experimental autoimmune encephalomyelitis-induced mice, by a decrease in the inflammatory cytokine interferon-gamma and by secretion of the anti-inflammatory cytokine IL-10 in response to the autoantigen MBP.
Subject(s)
Adjuvants, Immunologic/administration & dosage , Central Nervous System/pathology , Encephalomyelitis, Autoimmune, Experimental/prevention & control , Peptides/immunology , Th2 Cells/cytology , Adoptive Transfer , Animals , Central Nervous System/metabolism , Cytokines/metabolism , Female , Fluorescent Dyes , Glatiramer Acetate , Mice , Mice, Inbred BALB C , Muramidase/administration & dosageABSTRACT
The group of subtype I transmembrane tyrosine kinases includes the epidermal growth factor (EGF) receptor (ErbB-1), an orphan receptor (ErbB-2), and two receptors for the Neu differentiation factor (NDF/heregulin), namely: ErbB-3 and ErbB-4. Here we addressed the distinct functions of the two NDF receptors by using an immunological approach. Two sets of monoclonal antibodies (mAbs) to ErbB-3 and ErbB-4 were generated through immunization with recombinant ectodomains of the corresponding receptors that were fused to immunoglobulin. We found that the shared ligand binds to highly immunogenic, but immunologically distinct sites of ErbB-3 and ErbB-4. NDF receptors differed also in their kinase activities; whereas the catalytic activity of ErbB-4 was activable by mAbs, ErbB-3 underwent no activation by mAbs in living cells. Likewise, down-regulation of ErbB-4, but not ErbB-3, was induced by certain mAbs. By using the generated mAbs, we found that the major NDF receptor on mammary epithelial cells is a heterodimer of ErbB-3 with ErbB-2, whereas an ErbB-1/ErbB-2 heterodimer, or an ErbB-1 homodimer, is the predominant species that binds EGF. Consistent with ErbB-2 being a shared receptor subunit, its tyrosine phosphorylation was increased by both heterologous ligands and it mediated a trans-inhibitory effect of NDF on EGF binding. Last, we show that the effect of NDF on differentiation of breast tumor cells can be mimicked by anti-ErbB-4 antibodies, but not by mAbs to ErbB-3. Nevertheless, an ErbB-3-specific mAb partially inhibited the effect of NDF on cellular differentiation. These results suggest that homodimers of ErbB-4 are biologically active, but heterodimerization of the kinase-defective ErbB-3, probably with ErbB-2, is essential for transmission of NDF signals through ErbB-3.
Subject(s)
Antibodies, Monoclonal , ErbB Receptors/metabolism , Proto-Oncogene Proteins/metabolism , Animals , Antibodies, Monoclonal/pharmacology , Antibody Specificity , Base Sequence , Breast Neoplasms , CHO Cells , Cell Differentiation/physiology , Clone Cells , Cricetinae , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/analysis , Cyclins/biosynthesis , DNA Primers , Epidermal Growth Factor/metabolism , ErbB Receptors/biosynthesis , ErbB Receptors/immunology , Female , Glycoproteins/metabolism , Humans , Hybridomas , Immunohistochemistry , Intercellular Adhesion Molecule-1/analysis , Intercellular Adhesion Molecule-1/biosynthesis , Macromolecular Substances , Mammals , Mice , Mice, Inbred BALB C/immunology , Molecular Sequence Data , Neuregulins , Phosphorylation , Phosphotyrosine/analysis , Proto-Oncogene Proteins/biosynthesis , Proto-Oncogene Proteins/immunology , Receptor, ErbB-3 , Receptor, ErbB-4 , Restriction Mapping , Transfection , Tumor Cells, CulturedABSTRACT
The binding of the stem cell factor (SCF) to the c-kit-encoded receptor tyrosine kinase stimulates a variety of biochemical responses that culminate in cellular proliferation, migration, or survival. The extracellular domain of p145kit consists of five immunoglobulin-like domains. To confine the ligand binding site to this portion of the receptor we generated a panel of murine monoclonal antibodies (mAbs) to the Kit protein and identified two mAbs that efficiently displaced receptor-bound SCF and also inhibited proliferation of SCF-dependent human megakaryocytes. To map the epitopes of these mAbs we constructed and expressed soluble portions of the extracellular domain of Kit, which included either the two amino-terminal Ig-like domains (denoted Kit 1-2), three Ig-like domains (Kit 1-2-3), or the entire extracellular portion (Kit-X). All three recombinant proteins were recognized by the ligand inhibitory mAbs, suggesting that the SCF binding site resides in the amino-terminal half of the ecto-domain. Consistent with this conclusion, all of the soluble proteins inhibited SCF binding to Kit-expressing cells, and they also underwent specific covalent cross-linking to the radiolabeled ligand. However, whereas Kit 1-2-3 and Kit-X displayed comparable ligand affinities, deletion of the third Ig-like domain, in Kit 1-2, involved significant reduction in SCF binding. Hence, the binding site of SCF probably includes Ig-like domains 1 and 2, but structural determinants distal to this portion may also participate in ligand recognition.
Subject(s)
Antibodies, Monoclonal , Hematopoietic Cell Growth Factors/metabolism , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Receptors, Colony-Stimulating Factor/metabolism , Animals , Binding Sites , CHO Cells , Cricetinae , Cross-Linking Reagents , Mutation , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/immunology , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/immunology , Proto-Oncogene Proteins c-kit , Receptors, Colony-Stimulating Factor/genetics , Receptors, Colony-Stimulating Factor/immunology , Recombinant Proteins/metabolism , Sequence Deletion , Solubility , Stem Cell FactorABSTRACT
A novel technique was designed to conveniently determine substrate phosphorylation by tyrosine kinase. The technique is based on quantitation of phosphotyrosine content of the phosphoproteins, generated during the enzyme reaction, by radioimmunoassay. Here, we utilized high-titer monoclonal antibodies to phosphotyrosine, and radioiodinated bovine serum albumin-phosphotyrosine conjugate. The radiolabeled antigen was displaced from the complex formed in the assay by unlabeled phosphotyrosine, phosphotyrosine derivatives or phosphotyrosine-containing protein substrates. Half-maximal displacement was achieved at 0.4 +/- 0.05 microM by free phosphotyrosine, and at 40 +/- 3 and 45 +/- 4 nM by acetyl-phosphotyrosine and acetyl-phosphotyrosyl-glycine ethyl ester, respectively. Neither phosphoserine, phosphothreonine nor ATP cross-reacted with the phosphotyrosine antibodies. None of the components of the enzyme reaction interfered in the RIA. The method allows quantitation of the incorporated phosphate into tyrosyl residues without interference of serine/threonine phosphorylation. This technique avoids the use of short-lived [gamma-32P]ATP and omits the separation of the phosphorylated substrate from excess nucleotide.
Subject(s)
Protein-Tyrosine Kinases/chemistry , Radioimmunoassay , Tyrosine/analogs & derivatives , Animals , Antibodies/analysis , Kinetics , Liver/chemistry , Peptides/chemistry , Phosphorylation , Phosphotyrosine , Proteins/chemistry , Radioimmunoassay/methods , Rats , Tyrosine/analysis , Tyrosine/immunologyABSTRACT
The ERBB2 (also called HER2, neu, and c-erbB-2) gene product, which encodes a growth factor receptor, was implicated in the malignancy of human adenocarcinomas. An antibody directed to the rat oncogenic receptor has been previously shown to have an antitumor effect in model systems. In an attempt to extend this observation to the protooncogenic human receptor and also to understand the underlying mechanism, we generated a panel of monoclonal antibodies specific to the extracellular portion of the ERBB2 protein. The effects of the antibodies on tumor growth were compared with their cellular and biochemical actions in vitro. Surprisingly, opposing in vivo effects were observed: although some antibodies almost completely inhibited the growth in athymic mice of transfected murine fibroblasts that overexpress Erbb-2, other antibodies either accelerated tumor growth or resulted in intermediate responses. When tested on cultured human breast carcinoma cells or ERBB2 transfectants, the tumor-stimulatory antibody was found to induce significant elevation of tyrosine phosphorylation of the ERBB2 protein. In contrast, only partial correlation was observed between the capacity to restrict tumor growth and the effects of the antibodies on receptor degradation and cellular proliferation in vitro. This suggests that the antitumor antibodies affect both receptor function and host-tumor interactions. Our results may help establish experimental criteria for the selection of specific antibodies for use either alone or in conjunction with other molecules as pharmacological antitumor agents.
Subject(s)
Neoplasms, Experimental/pathology , Proto-Oncogene Proteins/physiology , Receptors, Cell Surface/physiology , Animals , Antibodies, Monoclonal , Cell Division , Humans , Mice , Mice, Nude , Neoplasm Transplantation , Oncogenes , Phosphoproteins/metabolism , Phosphotyrosine , Precipitin Tests , Protein-Tyrosine Kinases/physiology , Receptor, ErbB-2 , Tumor Cells, Cultured , Tyrosine/analogs & derivatives , Tyrosine/metabolismABSTRACT
Soluble IL-6 receptor (IL-6-R) purified to homogeneity from normal human urine was used for immunization of mice and rabbits. Spleen cells derived from a mouse showing a high binding titer to IL-6-R in an inverted solid phase radioimmunoassay (IsRIA) and in a Western blotting analysis were fused to mouse myeloma cells. The hybridomas were screened by the IsRIA, and 30 positive clones were isolated and characterized. They were suitable for affinity purification of the IL-6-R and for its detection by Western blot analysis, by ELISA and by sandwich type sRIA. Most of them inhibited the binding of labeled IL-6-R to IL-6 in a solid phase RIA.
Subject(s)
Antibodies, Monoclonal , Interleukin-6/metabolism , Receptors, Immunologic/immunology , Animals , Antibodies, Monoclonal/isolation & purification , Binding, Competitive , Chromatography, Affinity , Enzyme-Linked Immunosorbent Assay , Humans , Hybridomas/immunology , Mice , Rabbits , Radioimmunoassay , Receptors, Immunologic/metabolism , Receptors, Interleukin-6 , SolubilityABSTRACT
In the current study, immunocytochemical typing of intermediate filaments was used for a differential diagnosis of human lung tumors from transthoracic fine-needle aspiration biopsies (TFNAB). The authors have compared the cytologic diagnosis of 53 lung cancer cases with the immunofluorescence patterns obtained using a panel of monoclonal antibodies, five of which (KG 8.13, KM 4.62, Ks B.17, KS 8.12, KK 8.60) react with specific cytokeratin polypeptides and one with vimentin (VIM 13.2). Only in six of 23 samples cytologically diagnosed as squamous cell carcinoma did the immunocytochemical typing of cytokeratins (ICTC) confirm the cytologic diagnosis. In seven cases some of the tumor cells stained positively with antibody Ks B.17 specific for simple epithelial keratin (No: 18), suggesting the presence of some cells of glandular origin. In ten additional cases the ICTC was in conflict with the cytologic diagnosis of squamous cell carcinoma (i.e., antibodies Ks 8.12 and KK 8.60 were negative, and antibody Ks B.17, positive) supporting a diagnosis of adenocarcinoma. In 14 of 18 cases cytologically diagnosed as adenocarcinoma, the ICTC confirmed the diagnosis whereas in four cases additional presence of some squamous cells was noticed. The ICTC labeling of cases cytologically diagnosed as undifferentiated and large cell carcinomas was similar to that of the group of adenocarcinomas. Thus, the application of cytokeratin typing for TFNAB samples seems to provide a vital complementation to routine cytologic study, especially for cases cytologically diagnosed as squamous carcinoma.
Subject(s)
Adenocarcinoma/diagnosis , Antibodies, Monoclonal , Carcinoma, Squamous Cell/diagnosis , Carcinoma/diagnosis , Lung Neoplasms/diagnosis , Adenocarcinoma/pathology , Antibodies, Monoclonal/immunology , Biopsy, Needle , Carcinoma/pathology , Carcinoma, Squamous Cell/pathology , Diagnosis, Differential , Female , Fluorescent Antibody Technique , Humans , Immunohistochemistry , Intermediate Filaments/immunology , Lung Neoplasms/pathology , Male , Smoking/pathologyABSTRACT
Immunological cross-reactivity between tumor necrosis factor (TNF) binding proteins which are present in human urine (designated TBPI and TBPII) and two molecular species of the cell surface receptors for TNF is demonstrated. The two TNF receptors are shown to be immunologically distinct, to differ in molecular weight (58,000 and 73,000), and to be expressed differentially in different cells. It is further shown that polyclonal antibodies against one of the TNF binding proteins (TBPI) display, by virtue of their ability to bind the TNF receptor, activities which are very similar to those of TNF. These antibodies are cytotoxic to cells which are sensitive to TNF toxicity, induce resistance to TNF toxicity, enhance the incorporation of thymidine into normal fibroblasts, inhibit the growth of chlamydiae, and induce the synthesis of prostaglandin E2. Monovalent F(ab) fragments of the polyclonal antibodies lack TNF-like activities, but acquire them upon cross-linking with anti-F(ab)2 antibodies, suggesting that the ability of the anti-TBPI antibodies to mimic TNF correlates with their ability to cross-link the TNF receptors. This notion was further supported by data obtained in a comparative study of the TNF-like cytotoxicity of a panel of monoclonal antibodies against TBPI. The induction of TNF-like effects by antibodies to a TNF receptor suggests that TNF is not directly involved in intracellular signalling. Rather, it is the receptors to this cytokine which, when properly triggered in a process which appears to involve clustering of these receptors, transduce the signal for response to TNF into the cell's interior.
Subject(s)
Receptors, Cell Surface/metabolism , Tumor Necrosis Factor-alpha/metabolism , Animals , Antibodies, Monoclonal , Cell Line , Cells, Cultured , Cross Reactions , Epitopes/analysis , Female , HeLa Cells/metabolism , Humans , Immunoglobulin Fab Fragments , Mice , Mice, Inbred BALB C/immunology , Molecular Weight , Rabbits/immunology , Receptors, Cell Surface/immunology , Receptors, Tumor Necrosis Factor , Recombinant Proteins/metabolism , Skin/metabolismABSTRACT
Affinity chromatography of crude human urinary proteins on either human recombinant interleukin-6 (rIL-6) or human recombinant interferon-gamma (rIFN-gamma) or anti IFN-gamma receptor (IFN-gamma-R) monoclonal antibodies (McAb) yielded the two respective soluble receptors in significant amounts. A single sequence of 30 amino acid residues was obtained by N-terminal microsequencing of the protein peak purified in tandem by affinity chromatography on an IL-6 column and reversed-phase high-performance liquid chromatography. This sequence was identical with the predicted N-terminal sequence of IL-6-R as previously reported. The purified IL-6-R retained its biological activity. It was used for the preparation of specific anti IL-6-R monoclonal antibodies. Analysis of the eluted proteins from both IFN-gamma and anti IFN-gamma-R columns by inhibition of solid-phase radioimmunoassay, enzyme-linked immunosorbent assay, sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting proved the existence of soluble IFN-gamma-R in normal urine. This finding together with the already known presence of soluble TNF receptors and a soluble IL-2 receptor found both in plasma and in urine indicates that release of soluble cytokine receptors into body fluids is a general phenomenon which occurs under normal physiological conditions.
Subject(s)
Interleukin-6/urine , Receptors, Immunologic/urine , Chromatography, Affinity , Electrophoresis, Polyacrylamide Gel , Humans , Hybridomas/analysis , Immunochemistry , Interferon-gamma/urine , Proteinuria/urine , Radioimmunoassay , Receptors, Interferon , Receptors, Interleukin-6 , Recombinant Proteins/analysisABSTRACT
A human ovarian Brenner tumor presenting a wide spectrum of benign and malignant histologic features was studied for its patterns of intermediate filament expression. All epithelial elements of the tumor, regardless of their morphologic type, contained cytokeratins as their only intermediate filament component. Differences were detected, however, between tumor nests that displayed transitional epithelium and those with squamoid features. These differences were manifested by the presence of cytokeratin 18, in the former type only, and by the abundance of cytokeratins 10/11 in the latter. We also detected mixed epithelial nests in which both features were present, suggesting that the transitional epithelium transforms in polar fashion into squamous epithelium. Examination of cytokeratin patterns found in urothelium and in the surface epithelium of the ovary pointed to certain differences from the Brenner tumor epithelia. The significance of these latter findings with regard to cellular transformation and histogenesis of the Brenner tumor are discussed.
Subject(s)
Brenner Tumor/analysis , Cytoskeletal Proteins , Keratins/analysis , Ovarian Neoplasms/analysis , Antibodies, Monoclonal , Brenner Tumor/pathology , Desmin/analysis , Desmoplakins , Desmosomes , Epithelium/analysis , Female , Fluorescent Antibody Technique , Humans , Membrane Glycoproteins/analysis , Middle Aged , Ovarian Neoplasms/pathology , Ovary/analysis , Ovary/cytology , Urinary Bladder Neoplasms/analysis , Vimentin/analysisABSTRACT
Immunofluorescent and immunoperoxidase labelling of normal and metaplastic human submandibular salivary glands with a battery of cytokeratin-specific monoclonal antibodies was carried out. Labelling with a broad spectrum cytokeratin antibody (KG 8.13), as well as with antibody to cytokeratin polypeptide No. 18 (Ks 18.18) was observed in all the epithelial elements of the gland. Polypeptide No. 19, however, was present in ductal cells only, sparing the acini and the associated myoepithelium. Antibody KS 8.58, specific for cytokeratins Nos. 13 and 16, selectively labelled basal cells along the large ducts. Examination of squamous metaplasia associated with chronic suppurative sialadenitis indicated that the metaplastic cells display the same cytokeratin profile as the normal ductal cells and are not labelled with antibodies KS 8.58 and KK 8.60 which usually stain normal and pathological stratified epithelia. The significance of these observations for the histogenesis of normal salivary glands, as well as for the development of the metaplastic process, is discussed.
Subject(s)
Keratins/analysis , Salivary Gland Diseases/metabolism , Sialadenitis/metabolism , Submandibular Gland/analysis , Antibodies, Monoclonal , Child , Epithelium/analysis , Fluorescent Antibody Technique , Humans , Immunoenzyme Techniques , Metaplasia , Microscopy, Fluorescence , Peptides/analysis , Submandibular Gland Diseases/metabolismABSTRACT
The present study was directed towards the characterization of cell-specific histogenetic markers for the various epithelial elements of the adult and the developing guinea pig submandibular salivary gland. We have employed immunofluorescent labelling using three cytokeratin monoclonal antibodies, for which the polypeptide specificities towards guinea pig cytokeratins were determined. All the epithelial elements of the adult gland were positively labelled with two monoclonal antibodies, namely KG 8.13 ('broad spectrum' anti-cytokeratin) and antibody Ks B.18 (reactive with a simple cytokeratin-specific polypeptide of 49 X 10(3) Mr). Antibody KS 8.58 (reactive with a guinea pig cytokeratin polypeptide of 50 X 10(3) Mr) labelled the basal cells of the large ducts, as well as the myoepithelium. During development of the gland, the submandibular anlage and its primary and secondary branches with their terminal buds, were uniformly labelled with the three antibodies; however, the cytokeratin polypeptides reactive with antibody KS 8.58, which were apparently expressed in all cells of the developing ducts, gradually disappear from most of the ductal cells, starting at about 6 weeks of gestation, and remain only in the basal or reserve cells of the large ducts and the myoepithelium. These observations support the notion that the basal cells retain at least some of the properties of the embryonic glandular epithelium and could be considered as pluripotent reserve cells which may function as progenitors for other epithelial elements in the salivary glands epithelia.
Subject(s)
Embryonic and Fetal Development , Keratins/metabolism , Submandibular Gland/growth & development , Animals , Antibodies, Monoclonal , Antibody Specificity , Guinea Pigs , Immunohistochemistry , Keratins/immunology , Microscopy, Fluorescence , Peptides/metabolism , Submandibular Gland/embryology , Tissue DistributionABSTRACT
Immunofluorescent labeling of human salivary glands was carried out with a battery of monoclonal antibodies reactive with specific cytokeratin polypeptides. All the epithelial elements of the glands were positively labelled by a broad-spectrum cytokeratin antibody (KG 8.13) and by antibody Ks 18.18, which reacts with cytokeratin No. 18 exclusively. Labelling of frozen sections with antibody KM 4.62, which is reactive with the 40 Kd (No. 19) cytokeratin, was confined to the ductal system and apparently absent from the acini. Antibody KA-1, reactive with polypeptides 4, 5 and 6 stained both the myoepithelial cells and the basal cells of the large ducts. Antibody KS 8.58, however, reacted with the basal cells exclusively. It is thus proposed that the combined use of the various monoclonal antibodies may provide a most useful probe in studies on epithelial cell diversity in normal salivary glands as well as in pathological disorders of that gland.
Subject(s)
Keratins/metabolism , Peptides/metabolism , Salivary Glands/metabolism , Child , Child, Preschool , Epithelial Cells , Epithelium/metabolism , Histocytochemistry , Humans , Immunochemistry , Immunologic Techniques , Microscopy, Fluorescence , Salivary Glands/cytologyABSTRACT
The expression of cytokeratin polypeptides in squamous metaplasia of the human uterine cervix was investigated by immunocytochemical labeling with polypeptide-specific antibodies against cytokeratins. Immunofluorescence microscopic examination of cervical tissues using various monoclonal antibodies indicated that squamous cervical metaplasia expresses a unique set of cytokeratin polypeptides, this being distinctively different from that expressed by all of the normal epithelial elements of the exo- and endocervix. The development of metaplastic foci was accompanied by the expression of cytokeratin polypeptide no. 13, which is commonly detected in stratified epithelia, and by a reduction in the level of polypeptide no. 18, which is typical of simple epithelia. The 40-kilodalton cytokeratin (no. 19) described by Moll et al., which is abundant in the columnar and reserve cells of the endocervix, was found throughout the metaplastic lesions. Only in 'well-differentiated' metaplasias did we detect polarity of cytokeratin expression reminiscent of the staining patterns in the exocervix. This was manifested by the exclusive labeling of the basal cell layer(s) with antibodies KB 8.37 and KM 4.62, which stain the basal cells of the exocervix. Furthermore, a comparison of cervical metaplasia with squamous areas occurring within endometrial adenocarcinomas pointed to a close similarity in the cytokeratin expression of the two. We discuss the use of cytokeratins as specific markers of squamous differentiation, the relationships between squamous metaplasia and cervical neoplasia, and the involvement of reserve cells in the metaplastic process.
Subject(s)
Cervix Uteri/pathology , Keratins/analysis , Adult , Aged , Antibodies, Monoclonal , Cervix Uteri/cytology , Cervix Uteri/metabolism , Collodion , Electrophoresis, Polyacrylamide Gel , Epithelium/analysis , Female , Fluorescent Antibody Technique , Humans , Keratins/immunology , Keratins/physiology , Metaplasia/diagnosis , Metaplasia/metabolism , Metaplasia/pathology , Microscopy, Fluorescence , Middle Aged , Peptides/analysis , Peptides/immunology , Uterine Cervical Neoplasms/physiopathologyABSTRACT
We describe here antigenic cross-reactivity between the human 40-kilodalton cytokeratin polypeptide [Moll et al] and components of bovine desmosomal plaque, namely desmoplakins I and II. This relationship was revealed by an antibody (KM 4.62), raised against cytoskeletal preparation of cultured human breast adenocarcinoma cells (MCF-7) and selected by immunoblotting and immunofluorescent labeling. In cultured human cells that contain the 40-kD cytokeratin, antibody KM 4.62 labeled arrays of filaments throughout the cytoplasm. This antibody labeled the basal layer of nonkeratinizing squamous epithelia as well as various simple (normal and malignant) epithelia and epithelial elements of the thymus. In liver tissue, labeling was obtained only in bile ducts and canaliculi but not in the hepatocytes. In bovine cells and tissues, on the other hand, immunofluorescent labeling with antibody KM 4.62 was strictly confined to desmosomes. This was verified by double immunolabeling with both antibody KM 4.62 and specific cytokeratin or desmosomal antibodies. Immunoblotting analysis indicated that the former antibody reacts specifically with the high molecular weight components of the bovine desmosomal plaque, namely desmoplakins I and II. These immunochemical results suggest that bovine desmoplakins share same structural relationship with the human acidic, 40-kD cytokeratin.
Subject(s)
Cytoskeletal Proteins , Keratins/analysis , Membrane Proteins/analysis , Animals , Antibodies, Monoclonal , Cattle , Cell Line , Cross Reactions , Desmoplakins , Epitopes/analysis , Female , Fluorescent Antibody Technique , Humans , Keratins/immunology , Membrane Proteins/immunology , Molecular Weight , Neoplasms/analysisABSTRACT
We determined the reactivity of two monoclonal antibodies to cytokeratins that are typically expressed in certain stratified epithelia and several human squamous cell carcinomas using immunoblotting techniques and immunofluorescence microscopy. Antibody KS 8.12 reacted specifically with cytokeratin polypeptides nos. 13 and 16, and stained noncornified squamous epithelia in a rather uniform way. The examination of diverse human carcinomas showed all squamous cell carcinomas to be positively stained with this antibody, whereas all adenocarcinomas were negative. Another antibody, KK 8.60, reacted with polypeptides nos. 10 and 11, and uniformly stained the suprabasal layers of the epidermis. In several noncornified squamous epithelia (e.g., tongue, exocervix), in thymus reticulum epithelial cells, and in moderately and well differentiated squamous cell carcinomas this antibody exhibited a nonuniform labeling pattern that allowed the detection of individual cytokeratin-10/11-positive cells scattered throughout the tissue. It is concluded that antibodies KS 8.12 and KK 8.60 represent specific molecular probes for the definition of certain stages of squamous differentiation in normal development as well as in pathological processes such as squamous metaplasia and carcinogenesis. We propose the use of these antibodies in the differential diagnosis of carcinomas and their metastases.
Subject(s)
Carcinoma, Squamous Cell/pathology , Epithelial Cells , Keratins/analysis , Adenocarcinoma/pathology , Antibodies, Monoclonal , Antigen-Antibody Complex/analysis , Female , Fluorescent Antibody Technique , Humans , Skin/cytology , Tissue DistributionABSTRACT
In patients with rheumatoid arthritis (RA) a significantly decreased autologous mixed lymphocyte reaction (AMLR) was observed which varied widely and did not correlate with disease activity, clinical course or treatment schedules. When supernatants of AMLR cultures were tested for the presence of soluble factors no differences were found in regard to the suppression of allogeneic and mitogen induced lymphocyte proliferation. Furthermore both test groups failed to produce detectable amounts of interferon during the course of the AMLR, in contrast to the allogeneic situation were both RA patients and normal controls exhibited a similar interferon activity in the culture supernatants.