Keeping the fat on the right spot prevents contour deformity in temporalis muscle transposition.

The temporalis muscle transposition is a reliable, one-stage reanimation technique for longstanding facial paralysis. In the variation described by Rubin, the muscle is released from the temporal bone and folded over the zygomatic arch towards the modiolus. This results in unsightly temporal hollowing and zygomatic bulging. We present a modification of this technique, which preserves the temporal fat pad in its anatomical location as well as conceals temporal hollowing and prevents zygomatic bulging. The data of 23 patients treated with this modification were analysed. May classification was used for evaluation of mouth reanimation. Experts and patients scored visibility of the contour deformity on a 100-mm visual analogue scale (VAS) (score 0 = poor/100 = best). 3D images of the face were used to measure temporal hollowing and zygomatic bulging. 3D images were compared to those of controls with a similar gender and age distribution. After a median follow-up of 5.7 years, all patients achieved symmetry at rest. Eleven patients achieved symmetry while smiling with closed lips (May classification "Good"). A median (interquartile range [IQR]) VAS score of 19 (6; 41) was given by experts and 25 (5; 59) by patients themselves. 3D volumes of zygomatic bulging differed from those of control subjects, although all volume differences were small (median <3.3 ml) and temporal hollowing did not differ significantly. On the basis of our results, we conclude that our modified Rubin temporalis transposition technique provides an elegant way to conceal bulging over the zygomatic arch and prevents temporal hollowing, without the need for fascial extensions to reach the modiolus.

Paralytic ectropion treatment with lateral periosteal flap canthoplasty and introduction of the ectropion severity score.

BACKGROUND: Paralytic ectropion patients suffer from impairment of function and appearance of the lower eyelid and are at high risk of developing an exposure keratitis. A canthoplasty procedure can reduce the horizontal eyelid laxity and elevate the lower eyelid. We used a periosteal flap from the outer orbit to create a new canthal ligament. This study investigates the long-term outcomes of this technique. METHODS: Cross-sectional outcome study in which 30 cases of paralytic ectropion are treated with a lateral periosteal flap canthoplasty after adequate eyelid shortening. At the desired canthal height, a periosteal flap from the outer temporal orbital rim is mobilized around the rim and sutured in a double-breasted fashion to a tarsal strip. Effect of the operation is measured by comparing preoperative and postoperative photographs for signs of ectropion. For this purpose, a new photograph-based scoring method [the Ectropion Severity Score (ESS)] was developed and evaluated. RESULTS: The ESS proved to be reliable and sensitive to the presence of ectropion. Significant improvement of the ectropion sequelae was measured after a mean follow-up period of 2 years. In 3 cases (13%), a revision procedure was necessary because of relapse of lower eyelid sagging after a mean time of 1.9 years. In these cases, the periosteal flap could be reused. CONCLUSIONS: The ESS is a useful instrument to score the severity of paralytic ectropion. The periosteal flap canthoplasty is an effective procedure, with durable results in paralytic ectropion patients. The same periosteal flap can be used in a revision procedure.

Fluorescent location of malignant cells in fine needle aspirates.

Malignant cells in fine needle aspirates possess a cell surface protease which can be targeted with fluorescent affinity probes. Cells with active GB exhibit cell surface fluorescence when stained with such affinity probes. The nuclei of all cells on the slides can be counterstained with a nuclear fluorescent stain. Malignant cells are then located by their cell surface fluorescence and their diagnosis confirmed by examining their fluorescent nuclei. Normal cells and benign cells exhibit no cell surface fluorescence and can be ignored. This technique can be used to rapidly select cells of cytological interest in FNA samples obtained routinely and might be adapted for automated screening of FNA.

Evidence for the induction of a tumour associated cell surface protease on cytologically normal epithelial cells present in the sputum of patients possessing lung tumours.

Sputum obtained from healthy subjects and patients with known lung tumours has been challenged with fluorescent probes for the presence of an active cell surface protease. The mature epithelial cells from healthy patients' sputum lacked ability to bind these fluorescent probes whilst the majority of mature epithelial cells in the tumour patients' sputum bound these probes and consequently fluoresced. This demonstrable difference in the cell surface chemistry of mature epithelial cells was linked to the presence of lung tumour cells, which also possessed this cell surface protease. The mechanism of this induced cell surface enzyme appearance is not understood.

Fluorescent location of lung tumour cells.

Cells were collected on glass slides by touching tumour surfaces (A) and normal regions (B) of the lung. The slides were stained with a nuclear stain and a fluorescent probe for a tumour associated cell surface protein. The (B) slides from the normal regions lacked fluorescent epithelial cells. The tumour slides (A) contained typical tumour cells and dyskaryotic cells which exhibited cell surface fluorescence.

Sputum cells from lung tumor patients carry a cell surface marker not found in normal sputum.

Cells were collected from sites of known lung tumours and corresponding control areas of these lungs. Fluorescent staining demonstrated that the tumour cells and epithelial cells (cytologically these cells appeared normal) both possessed a receptor for these fluorescent probes. Fluorescent labelling of sputum cells from these tumour patients also resulted in fluorescent labelling of these "cyto logically normal" epithelial cells. No such fluorescent epithelial cells were observed in sputum samples collected from control subjects or in cells collected from the control areas of the tumour patients' lungs. We conclude that a cell surface protein receptor is expressed in lung tumour-associated epithelial cells but is absent from control sputum epithelial cells.

On the stability of a tumour cell surface protease after exposure to 6N HCl.

Well defined acinar tumour cells in frozen sections were exposed to 6NHCl for 5 h at room temperature. A technique was designed to monitor the activity of the enzyme, guanidinobenzoatase (GB), on these tumour cells; this involved cross-linking the enzyme to the cell surface and challenging the active centre with known fluorescent probes which only bind to the functional enzyme. It was demonstrated that the enzymic activity can be regained by appropriate folding of the protein.

A simple clinical method for the preparation of improved cervical smears-approximating to monolayers.

Conventionally prepared cervical smears contain multilayers of cells deposited within strands of mucin. The present study is concerned with chemical reduction of disulphide bonds in the mucin leading to depolymerisation prior to forming a smear in the conventional manner. The resultant distribution of cells on the slide is similar to that obtained by machines designed to produce monolayers of cells. These monolayers have been developed for use in automated analysis of cervical smears and sputum samples. This new technique does not interfere with conventional PAP analysis of dyskaryotic cells nor does it interfere with the fluorescent location of such cells of cytological interest.

Fluorescent location of cells of cytological interest in cervical smears prestained with thionin.

Cervical cells of cytological interest were located in conventional smears, treated with thionin for quantitative DNA staining by subsequent treatment with fluorescent probes for a cell surface protease. Normal mature cervical epithelial cells failed to bind these fluorescent probes whilst metaplastic cells, glandular cells, and dyskaryotic cells were readily located. By this means, the nuclear staining of these fluorescent cells of cyotological interest enabled them to be classified by a cytologist.

Correlation of cell surface fluorescence with conventional PAP analysis of cells of cytological interest obtained from cervical scrapes.

Archival PAP stained cervical smears were destained and treated with a fluorescent probe for a cell surface enzyme (GB). Cells which exhibited cell surface fluorescence were demonstrated to be cells of cytological interest in the analysis of cervical smears. These cells could be directly related to PAP and reclassified by subsequent restaining with PAP. Fluorescent cell surface technology was shown to be compatible with conventional PAP staining.

Selectivity of the plasminogen activator inhibitor (PAI-1) for the iso enzyme of guanidinobenzoatase on the surface of colonic carcinoma cells.

The interaction of plasminogen activator-inhibitor (PAI-1) with a cell surface protease, guanidinobenzoatase (GB), has been studied in free solution and on the surface of colonic epithelial cells. It has been demonstrated that PAI-1 recognises and inhibits the iso enzymic form of GB associated with colonic carcinoma cells but fails to bind to the iso enzymic form of GB associated with normal donor colonic epithelial cells. This interaction is mediated by a lysyl binding site on the GB: complex formation prevents GB binding to fibrin fibrils which also involves lysyl binding sites.

Differential competitive inhibition: the theory of a concept.

The concept of differential competitive inhibition of cell surface isoenzymes is discussed. If the isoenzymes have different structures and functions it is likely that they will handle active site directed molecules at different rates. This could be tested by employing two competitive inhibitors in sequence and exploited if the second inhibitor is fluorescent. Two practical examples of this technique will be presented in the following manuscripts.

The fluorescent location of abnormal cervical cells employing the principle of differential competitive inhibition.

The screening of cervical smears is concerned with the detection of abnormal epithelial cells which may be indicative of cervical intraepithelial neoplasia (CIN). Several types of cells in cervical smears possess a cell surface protease where isoenzymic forms of this enzyme can be differentially inhibited. Using this phenomenon a simple fluorescent technique has been developed in conjunction with differential competitive inhibition which enables abnormal cervical epithelial cells to bind the fluorescent probe whilst other cells do not bind the probe. The abnormal cells can then be located by fluorescence microscopy and their co-ordinates recorded for subsequent characterisation of these cells by nuclear analysis employing haematoxylin to stain the nuclei.

Differential competitive inhibition of a cell surface protease on normal epithelial cells and carcinoma cells of the colon.

The cell surfaces of normal colonic epithelial cells and colonic carcinoma cells both possess a protease referred to as guanidinobenzoatase (GB). Previous studies have shown that these cells possess two distinct isoenzymic forms of GB which could be distinguished by their selective recognition of cytoplasmic protein inhibitors of GB. In the present study we have used competitive inhibitors of GB to demonstrate the differential inhibition of the GB on normal colonic epithelial cells whilst the GB on colonic carcinoma cell surfaces remains active. The enzymic status of GB on these cells has been determined by challenging the treated cells in frozen sections with a second fluorescent inhibitor, followed by fluorescence microscopic analysis.

Fluorescent location of malignant cells in smears obtained from sputum.

A fluorescent probe (rhodamine a-N-agmatine) has been used to locate cells possessing a surface protease in sputum smears. Malignant epithelial cells possess this protease and can be quickly located by this technique. These results have been confirmed by using a second fluorescent probe (9-animo acridine) for this same cell surface protease.

Retinoic acid inhibition of a tumour protease immobilised on cell surfaces and in free solution.

Retinoids are inhibitors of tumour cell proliferation in culture and have been shown to suppress carcinogenesis and decrease the levels of proteases. The present study has demonstrated that retinoic acid is a potential non-competitive inhibitor of a protease (GB) immobilised on the surfaces of tumour cells in thin sections and free GB in solution. The enzymic status of GB on the cell surfaces in sections has been determined by challenging the retinoic acid-treated cells with a second fluorescent inhibitor (9-AA), followed by fluorescence microscopic analysis. The inhibition of cell surface GB by retinoic acid was demonstrated to be reversible. The activity of soluble GB has been measured by the MUGB assay in the presence and absence of retinoic acid. It is suggested that retinoic acid acts on GB by interacting with a binding site, different from the active site, and causes major conformational changes, resulting in enzyme inhibition. It is possible that the modulation of GB activity by retinoic acid may play a role in the control of cell migration and metastasis.

Inhibition of a tumour protease with 3,4-dichloroisocoumarin, pentamidine-isethionate and guanidino derivatives.

Guanidinobenzoatase (GB) is a cell surface proteolytic enzyme capable of degrading fibronectin, and is associated with tumour cells and cells capable of migration. The location of active GB in sections has been demonstrated with 9-aminoacridine (9-AA), a competitive inhibitor of GB. 3,4-Dichloroisocoumarin (3,4-DCI) and pentamidine isethionate (PI) are inhibitors of trypsin-like enzymes. It has now been demonstrated that 3,4-DCI, PI, and guanidino derivative compounds are significant inhibitors of GB, on the surfaces of lung squamous cell carcinoma cells in frozen sections and free GB in solution. Dexamethasone acetate (DMA) and medroxy-progesterone (MP) did not show any significant inhibition of GB activity. These molecules lack a reactive chloride or guanidino groups and are thought to react at the nuclear level, rather than directly on this cell surface protease. Kinetic studies have shown that 3,4-DCI, PI and guanidino derivatives are reversible competitive inhibitors of GB, as determined in vitro on the purified enzyme. The inhibition resulting with 3,4-DCI was a time-dependent process. It is suggested that these inhibitors interact with GB by binding to its active site, resulting in the formation of enzyme-inhibiter complexes (GB-I). The GB-I complexes can be dissociated with SDS treatment, resulting in the regain of GB activity.

The interactions of protein inhibitors with tumour proteases studied in solution and immobilised on cell surfaces in frozen sections.

The cell surface protease guanidinobenzoatase (GB) has been purified from human colonic and lung carcinoma tissue by an affinity step involving the binding of the enzyme either onto fibrin fibrils or onto agmatine-sepharose. The inhibitor protein (I) was extracted from the cytoplasm of tumour cells and isolated by an affinity step involving the binding of I to GB on the surface of cultured carcinoma cells. The interaction of GB and I in solution was followed by kinetic studies employing the release of the fluorescent 4-methylumbelliferone (MU) from the synthetic substrate 4-methylumbelliferyl-p-guanidinobenzoate (MUGB). The interaction of soluble I with membrane bound GB was followed by using the yellow fluorescent probe 9-aminoacridine (9AA) which binds to active GB but not to GB-I. The results of these studies demonstrated the presence of isoenzymic froms of GB which were recognized specifically by their appropriate isoinhibitor, isolated from the appropriate cell type. This high degree of selectivity suggests a cell specific regulatory role for the inhibitors and the possibility that they might be used for the delivery of cytotoxic molecules to the surface of specific types of tumour cells.

Association and dissociation of a protease and its inhibitor on the surface of lung squamous cell carcinoma cells.

Squamous cell carcinoma cells possess a cell surface protease, referred to as guanidinobenzoatase (GB). GB is a plasminogen-activator-like enzyme which can be located by the fluorescent probe 9-amino acridine in frozen sections. Fluorescence microscopy has been used to study the inhibition of this GB, the displacement of inhibitor from GB, the displacement of GB from the cell surface receptor and the preparation of both active GB and inhibitor, obtained from these frozen sections of tumour tissue.