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Notes: The pattern of epithelial keratin expression in the normal anal canal has not been extensively defined and is a necessary prerequisite to the interpretation of alterations in these intermediate filaments in pathological anal epithelial lesions. Thirty-five frozen tissue specimens of resected haemorrhoids were investigated immunohistolo-gically for expression of 14 individual keratins (K) using a panel of 17 monoclonal antibodies. Perianal skin showed basal expression of karatinocyte Ks 5, 14 and 17, and suprabasal expression of keratinocyte Ks 14, 10, 1 and 16. Anal squamous epithelium showed persistent basal K5 and 17, basal and suprabasal K4, 13 and 16 positivity, with sporadic expression of K1 and 10. The expression of simple epithelial keratins in squamous epithelium adjacent to the anal transitional zone varied with basal expression of K7, K8, K18 and K19 and sporadic suprabasal expression of K7 and K19. The anal transitional zone (ATZ) expressed K19, as found in transitional epithelia elsewhere. The full thickness of epithelium was positive for the simple epithelial Ks 7, 8 18 and 19. Marked heterogeneity of keratinocyte keratin expression was seen. Basal layers expressed Ks 4, 13, 14 and 17 and variably K16, while suprabasal layers expressed Ks4 and 13, 14 and 17 and variably K16, while suprabasal layers expressed Ks4 and 13 and variably K14, 16 and 17. This anomalous expression of keratinocyte K4 and 13 has also been documented in transitional epithelium of the bladder. The anal glands and ducts showed a keratin distribution similar to the transition zone. Rectal columnar epithelium expressed simple keratins 7, 8 18 and 19. In addition, low levels of keratinocyte keratins were found as indicated by heterogeneous staining for K4, 13, 14 and 16. The overall pattern, particularly in the region of the anal transitional zone and immediately adjacent squamous and columnar epithelia, is of a flexible epithelial cell population able to express a range of keratins unrestricted by a particular morphological phenotype. In the light of these results, analysis of changes in keratin distribution within anal carcinomas may assist classification by providing information on the state of differentiation and histogenesis of these tumours.

Notes: The expression patterns of basement membrane components and keratin intermediate filament proteins were studied in normal human bronchial epithelium and 56 lung carcinomas using monoclonal antibodies to laminin, type VII collagen and the individual keratins 14, 16, 17 and 18. In normal lung, laminin and type VII collagen were present between the epithelium and the lamina propria of bronchi and bronchioles. Keratin 14 was expressed in the basal cells, keratin 17 in the basal and some suprabasal cells and keratin 18 in the columnar cells of the bronchi and bronchioles. Keratin 16 was not present in normal bronchial epithelium. Laminin was found in all subtypes of lung carcinoma, but type VII collagen was present only in squamous cell carcinomas, where it showed a reduction in expression with decreasing differentiation. Type VII collagen was not identified in adenocarcinomas, small cell carcinomas or carcinoids. Antibodies to basal cell keratins 14 and 17 also displayed positivity only in squamous cell carcinomas, although no correlation with the degree of differentiation could be observed. Keratin 16 appeared to be a marker of the squamous phenotype, rather than of hyperproliferation. The keratin 18 marker for columnar epithelial cells showed a reaction pattern opposite to that of the basal cell keratins, being extensively present in adenocarcinomas, small cell carcinomas and carcinoids, with less expression in squamous cell carcinomas. This study shows a correlation between the presence of type VII collagen and the basal cell keratins 14 and 17, and a negative correlation between these components and keratin 18. These findings are likely to be useful in identifying lung cancer subtypes.

Notes: The characteristic expression of keratins by keratinocytes is well documented. A typical ‘hyperproliferative’ profile of epidermal keratin expression occurs in psoriasis, wound healing and warts. This study analyses keratin expression in cutaneous lichen planus to determine abnormalities of differentiation occuring in this inflammatory disorder. Using a panel of monoclonal antibodies 28 samples (20 patients) were studied. The results showed that squamous differentiation was unaffected, with keratins K1 and K10 being expressed normally for the site sampled. The main abnormalities included extension of reactivity of the basal cell marker, LH8, into the suprabasal compartment. Keratin K17, usually restricted to adnexal structures, was variably expressed in the basal and suprabasal layers of the interfollicular epithelium of affected epidermis. Keratins K6 and K16, found suprabasally in hyperproliferative states, were detected both basally and suprabasally in all diseased samples. The keratin profile in lichen planus is analogous to the wound healing response. Suprabasal keratin K17 is found in psoriasis, wound healing and viral warts so the changes in keratin K17 may reflect hyperproliferative changes. It is likely that the changes in epidermal keratin expression are due to up-regulation of specific keratin genes by the production of cytokines and inflammatory mediators from the lymphocytic infiltrate typical of lichen planus.

Notes: The distribution pattern of the basement membrane components type VII collagen and laminin, was studied immunohistochemically in normal human head and neck tissues and in a series of benign and malignant tumours from the same site. Using monoclonal antibodies, a basement membrane containing type VII collagen and laminin could be demonstrated beneath the epithelial cell layer in 16 normal head and neck tissues from different localizations. Unlike type VII collagen, laminin was also abundantly present around blood vessels and muscle fibres.With respect to 42 squamous cell carcinomas studied, type VII collagen and laminin were present in basement membranes surrounding small and large tumour fields, independent of the tumour grade. Type VII collagen was demonstrated in the cytoplasm of tumour cells in 36% of the cases, while the antibody to laminin displayed a basement membrane staining pattern mainly. Both antibodies showed a staining gradient in more than half of the cases, with strong staining in the centre of the tumour and weakening of the staining towards the tumour periphery.In a series of 22 salivary gland tumours consisting of 19 pleomorphic adenomas and three adenoid cystic carcinomas, the distribution pattern of type VII collagen and laminin was very heterogeneous. Laminin was present in 17 and type VII collagen in 10 of 19 cases of pleomorphic adenoma, mostly scattered throughout the tumour fields. In the tumours positive for type VII collagen areas with little or no positivity were also found. A correlation between type VII collagen positivity and the presence of basal cell keratin 14 positivity was noticed in the majority of cases. The three adenoid cystic carcinomas studied displayed random positivity with the laminin antibody, whilst the antibody to type VII collagen showed little positivity in these cases.

Notes: In this Study a variety of immunoelectron microscopic methods were used to define the precise ultrastructural binding site of epidermolysis bullosa acquisita antibodies (EBA-Ab). We used two EBA sera which immunoblotted with the same skin-extracted protein as that labelled by a monoclonal antibody (LH7.2) which is known to react with the carboxy terminus of type VII collagen. Gold-conjugated antibodies were used in two different immunoelectron microscopic procedures to compare the labelling characteristics of EBA-Ab and LH7.2 in normal human skin. Antibody incubations were performed using ultra-thin cryosections of unfixed skin and thin slices of fresh skin (en bloc technique) before conventional fixation and embedding in Epon. Both methods showed similar labelling features for both EBA-Ab and LH7.2 With ultra-thin cryosections there was labelling of the lamina densa and an undefined component of the sub-lamina densa region. With the en bloc technique, labelling of dermal ends of anchoring fibrils and of amorphous material recently defined as‘anchoring plaques’was evident. There was no labelling of the central banded portions of anchoring fibrils. We conclude that EBA-Ag is localized to the dermal ends of anchoring fibrils in addition to the lamina densa and possibly anchoring plaques, and thus has the same distribution as the carboxy terminus of type VII collagen.

Notes: Topical antibiotics and antiseptics are widely used to prevent and treat bacterial infections in wounds and ulcers. Experimental data suggest that some antimicrobial agents may have an adverse effect on several aspects of the tissue repair process, including retardation of wound epithelialization. The aim of this study was to investigate the comparative cytotoxic effects of a range of antiseptics and antibiotics using human keratinocytes transformed by Simian virus 40 (SVK14 cells).SVK14 cells were grown to semi-confluence by adding 6 × 1014 cells per petri dish. After 48 h the cells were exposed to serial dilutions of the therapeutic concentrations (shown in parentheses) of each of the following agents; hydrogen peroxide (3%), cetrimide (1%), sodium hypochlorite (o·5%), povidone iodine (4%), neomycin (1%), bacitracin (50 units/ml), polymyxin B sulphate (10000 units/ml). The cells were exposed to the drug for 15 min and then washed and incubated in the culture medium (RPMI 1640 plus 10% foetal calf serum) for 24 h. Dead cells were then washed off and the viable adherent cells were trypsinized and counted in a Coulter counter.At therapeutic concentrations none of the antibiotics was found to be cytotoxic, whereas all of the antiseptics produced 100% killing of SVK14 cells. Dilutions of therapeutic concentrations of the antiseptics ranging between 1000- and 20000-fold (depending on the agent) were needed in order to achieve no-effect levels. Calculations based on 100% killing values for the antiseptics indicate that their order of toxicity from highest to lowest is: sodium hypochlorite, cetrimide, povidone iodine and hydrogen peroxide.Whilst it is appreciated that more work is needed in order to assess the exact relevance of these findings to clinical practice, it should be noted that SVK14 cells share many of the characteristics of normal keratinocytes and that these preliminary results are broadly in line with in vitro, in vivo and human volunteer findings made by other investigators studing antimicrobial toxicity profiles. We conclude that this cell line may be useful in studying the epithelial cytotoxicity of drugs in vitro and that care should be exercised in the selection of antimicrobial agents for use in wound management.

Notes: Frozen sections of punch biopsies from normal epidermis and psoriatic involved and uninvolved epidermis have been examined immunocytochemically using a panel of anti-keratin monoclonal antibodies with various specificities in the skin. Since psoriasis is thought to involve hyperproliferative expansion of the basal compartment from one to about three cell layers in thickness, the samples were screened with antibodies to intermediate filament determinants associated with basal cells, suprabasal cells and hyperproliferating keratinocyte-derived cell lines, respectively. The basal—suprabasal division was observed to be intact, with only one layer of basal cells demarcated by the specific antibodies used under all circumstances. this suggests that (a) psoriatic ‘basal cell hyperproliferation’ may not specifically involve the basal cell compartment containing the stem cells, but rather a population of amplifying transit cells which are predominantly suprabasal, and that (b) while keratinocyte differentiation begins as the cells lose contact with the basal lamina, the first stages at least of differentiation are not dependent on the loss of the capacity to divide.

Notes: A case of mepacrine pigmentation occurring in a patient with systemic lupus erythematosus has been investigated by fluorescent light microscopy, gas-liquid chromatography and analytical electron microscopy. There is strong evidence for the presence of mepacrine itself within the typical granules, which have been shown by electron microscopy to be membrane bound and intracellular. Analytical electron microscopy also showed that the granules contain large quantities of iron and smaller quantities of sulphur.