Anti-keratin Staining Research Articles

STAINING PATTERNS OF HUMAN PRE-MALIGNANT ORAL EPITHELIUM AND SQUAMOUS CELL CARCINOMAS BY MONOCLONAL ANTI-KERATIN ANTIBODIES

Formalin-fixed, paraffin-embedded biopsies of metaplastic keratinized oral mucosa (fibromas and leukoplakias), oral mucosa with epithelial dysplasia and oral squamous cell carcinomas were stained with two monoclonal anti-keratin antibodies (AE1 and AE2). Intense suprabasal staining was seen with AE1 in metaplastic keratinized epithelium, whereas staining of adjacent normal unkeratinized epithelium generally was restricted to basal cells. In dysplastic epithelium and squamous cell carcinomas, staining with AE1 revealed a highly disturbed anti-keratin staining pattern. AE2 stained metaplastic keratinized epithelium in a suprabasal pattern but adjacent unkeratinized epithelium did not stain. In dysplastic epithelium and squamous cell carcinomas, AE2 staining was variable and sometimes absent. Further studies are indicated to clarify whether changes in anti-keratin staining patterns can be used for diagnostic and prognostic purposes.

P.166 Salivary urea,creatinine,ascorbic and oxalic acid in CRF patients

To gain preliminary data on the location of a 195 kD cell peripheral protein of epidermal keratinocytes recognized by monoclonal antibody AE11, immunofluorescence staining was carried out on cultured human epidermal cells. B11 antidesmosomal polyclonal antibody and AE3 antikeratin monoclonal antibody staining were used as comparative reference controls. Desmosomal antigens span the cell membrane and keratins constitute a family of cytoplasmic proteins. The cultured cells were systematically used either unfixed, fixed with 4% paraformaldehyde, or fixed with methanol at 4°C. The former two preparations were designed to expose only the surface antigens, whereas the latter method permeated the membrane to allow cytoplasmic staining. Differences in staining patterns were observed in the basal layer as compared with the upper layers with all three antibodies. B11 antidesinosomal antibody staining was consistent in pattern after either paraformaldehyde or methanol fixation. In addition to the punctate staining along adjacent cell surfaces as shown in the basal layer, the surface planes of the upper cells exhibited parallel arrays of linear streaks, demonstrating the distribution of desmosomal proteins. Antikeratin staining by AE3 showed the typical filamentous staining in basal cells. However, a homogeneous patchy staining of suprabasal cells was observed. The presence of punctate surface staining using antikeratin antibody on paraformaldehyde fixed cells suggests leakage of keratin to the cell surface. AE11 showed stronger staining in the top cells on methanol treated cultures and a punctate surface staining of the cells fixed with paraformaldehyde. These observations provide useful preliminary information in localizing peripheral proteins in epidermal keratinocytes.

Alagille syndrome with a spontaneous appearance of the interlobular bile ducts

We report a rare case of a 10 year old girl diagnosed as having Alagille syndrome on the basis of a characteristic face, posterior embryotoxon, cholestasis, peripheral pulmonary artery stenosis and the absence of interlobular bile ducts in a liver biopsy at 1.5 years of age. Since 1.5 years old, she had been in good health without medication and the serum biochemical liver function tests indicated no progression of cholestasis. A second liver biopsy at 9.5 years of age showed normal interlobular bile ducts confirmed by anti-keratin staining at each of the five examined portal areas. Alagille syndrome is usually associated with the progressive disappearance of interlobular bile ducts. The findings of interlobular bile ducts in the second liver biopsy were therefore rare and unique to this case.

Micrometastases from squamous cell carcinoma in neck dissection specimens

The incidence of micrometastases in cervical lymph nodes from squamous cell carcinomas of the head and neck was studied using routine histopathological examination. Micrometastases were found in 66 lymph nodes in 41 of the 92 tumor-positive neck dissection specimens. The detection of these micrometastases influenced postoperative treatment in 3 of the 77 patients with neck node metastases. The value of additional sectioning for detecting micrometastases was thus assessed. Sectioning at a deeper level in 600 originally histopathologically negative lymph nodes from 64 patients revealed 7 additional micrometastases in 5 patients. Antikeratin staining with a mixture of two monoclonal antibodies (AE1 and AE3) revealed 4 micrometastases in 739 originally histopathologically negative lymph nodes in 3 of 13 patients studied. Because of the unknown prognostic significance of micrometastases and the consequent arbitrary consequences for postoperative treatment, present findings show that the extra workload of immunostaining and deeper sectioning does not warrant their routine use in clinical practise.

An Immunohistochemical Study

Proliferative vitreoretinopathy may be associated with taut subretinal membranes (SRMs) that prevent retinal reattachment. Light microscopic and immunohistochemical procedures were used to identify the cell composition of 15 surgically excised SRMs. Anti glial fibrillary acidic protein (anti-GFAP) and anti S-100 protein stainings were used to examine the distribution of glial cells, and antikeratin staining was used to investigate the distribution of epithelial cells. Our findings confirm the presence of retinal pigment epithelium (RPE) and glial cells in human SRMs. A semiquantitative evaluation of the immunohistochemical studies demonstrated the prevailing role of cells derived from the RPE (48.4%). The glial component of the SRMs appeared to be smaller than in epiretinal membranes, but was not negligible (30.8%). A high incidence (26.6%) of damaged external retina adhered to the SRMs was also observed.

Immunofluorescence Localization of Peripheral Proteins in Cultured Human Keratinocytes

To gain preliminary data on the location of a 195 kD cell peripheral protein of epidermal keratinocytes recognized by monoclonal antibody AE11, immunofluorescence staining was carried out on cultured human epidermal cells. B11 antidesmosomal polyclonal antibody and AE3 antikeratin monoclonal antibody staining were used as comparative reference controls. Desmosomal antigens span the cell membrane and keratins constitute a family of cytoplasmic proteins. The cultured cells were systematically used either unfixed, fixed with 4% paraformaldehyde, or fixed with methanol at 4 degrees C. The former two preparations were designed to expose only the surface antigens, whereas the latter method permeated the membrane to allow cytoplasmic staining. Differences in staining patterns were observed in the basal layer as compared with the upper layers with all three antibodies. B11 antidesmosomal antibody staining was consistent in pattern after either paraformaldehyde or methanol fixation. In addition to the punctate staining along adjacent cell surfaces as shown in the basal layer, the surface planes of the upper cells exhibited parallel arrays of linear streaks, demonstrating the distribution of desmosomal proteins. Antikeratin staining by AE3 showed the typical filamentous staining in basal cells. However, a homogeneous patchy staining of suprabasal cells was observed. The presence of punctate surface staining using antikeratin antibody on paraformaldehyde fixed cells suggests leakage of keratin to the cell surface. AE11 showed stronger staining in the top cells on methanol treated cultures and a punctate surface staining of the cells fixed with paraformaldehyde. These observations provide useful preliminary information in localizing peripheral proteins in epidermal keratinocytes.

Staining patterns of rodent squamous epithelia by monoclonal anti‐keratin antibodies

Anti-keratin staining patterns were examined by immunofluorescence in the epidermis, oral mucosa and forestomach of rats and mice by monoclonal antibodies AE1 and AE2. In contrast to human tissues enzymatic pretreatment of sections was necessary even when fresh-frozen tissue was used, suggesting masking of the antigens in vivo. AE1 stained the cytoplasm of spinous cells in most epithelia, whereas basal cell staining varied. AE2 showed suprabasal cytoplasmic staining in epidermis, forestomach and palate, whereas the other epithelia were stained only in keratohyalin granules and membranes of cornified cells. In some epithelia a small number of irregularly distributed basal cells stained positive with AE2, indicating heterogeneity in the basal cell compartment. Thus, the anti-keratin staining pattern varies both regionally and, as earlier shown in human epithelium, with the stage of maturation of cells within a given epithelium. The present study provides a basis for further studies of epithelial differentiation during normal and pathologic development.

Immunoperoxidase anti-keratin staining of epidermal and pilar cysts

Epidermal and pilar cysts have been stained with anti-keratin antibodies raised from human hair and human callus. The staining properties of the two cysts are different and, in general, support the idea that they originate from epidermis and trichilemmal hair follicle layer respectively.

Cell culture of mammalian thymic epithelial cells: Growth, structural, and antigenic properties

The thymus plays an important role in the maturation and differentiation of T lymphocytes. Many of its functions have been attributed to its epithelial component. Past in vitro studies of putative thymic epithelial cells have been hampered by the inability to produce well-characterized cultures of these cells. Using lethally irradiated 3T3 cells as a feeder layer, we have succeeded in growing virtually pure cultures of thymic epithelial (TE) cells from rabbits, mice, and humans. Antikeratin staining provides an unambiguous criterion for positive identification of the epithelial cells. These cells were found to lack Ia-like or theta-like antigens. The ability to culture large quantities of mammalian TE cells should allow for their detailed functional characterization.

Keratin filaments of corneal epithelial cells

Intermediate filaments with a diameter of approximately 8–10 nm are present in great abundance in the cytoplasm of corneal epithelial cells. In order to study the immunological relationship between these filaments and the tonofilaments of epidermal cells, we prepared an antiserum against keratin proteins isolated from the stratum corneum of human epidermis. Immunofluorescent staining of frozen sections of rabbit and human corneas with this anti-keratin antiserum resulted in the intense staining of the entire corneal epithelium, but no staining of the endothelium or any of the stromal components. Similar staining of cultured rabbit and human corneal epithelial cells revealed a cytoplasmic network of wavy fibers distinct from microfilaments and microtubles. Electron microscopic examination of cultured rabbit corneal epithelial cells showed that they formed a stratified squamous structure with prominent desmosomal cell-cell junctions and wavy bundles of cytoplasmic 8–10 nm filaments. These results suggest that the 8–10 nm filaments of corneal epithelium are immunologically related to tonofilaments of the epidermis and are composed of keratin proteins. Since corneal epithelium is the only keratin-containing cell type in normal cornea, anti-keratin staining provides a simple, specific and sensitive method for identifying corneal epithelial cells. Further characterization of the keratins synthesized by corneal epithelial cells under various in vivo and in vitro conditions may provide useful information concerning the mechanisms of corneal epithelial differentiation.